N,N-dimethyltryptamine (DMT) belongs to a class of drugs known as serotonergic psychedelics, which produce profound shifts in consciousness by dramatically altering perception and causing hallucinatory phenomena. In humans, DMT has a rapid onset and the effects, which include feelings of dissociation from external reality, entering into an ‘otherworldly dimension’, and intense internal visual imagery, typically last for less than 15 minutes. DMT has been used in ceremonial and medicinal settings across indigenous cultures for millennia in the form of the botanical brew ayahuasca, and it shows potential as a neuroplasticity promoting therapeutic. DMT also occurs endogenously in the mammalian brain and has been characterized as a putative neurotransmitter, but little is known about its function, endogenous regulatory mechanisms, or the mechanisms underlying its consciousness altering effects. We used rat models to study DMT biosynthesis and the neural correlates of administered DMT to advance mechanistic understanding of this endogenous psychedelic compound. A critical rate limiting step in DMT biosynthesis is the methylation of tryptamine by Indolethylamine-N¬-methyltransferase (INMT), which is hypothesized to be a necessary enzyme for endogenous DMT production. We tested this in INMT knockout rats and demonstrated that the absence of INMT does not affect tryptamine methylation, suggesting that INMT is not necessary for this process. In addition, we showed that recombinant rat INMT was inactive with tryptamine as a substrate. Since DMT has been demonstrated to occur in rat brain at levels comparable to other monoamine neurotransmitters, we conclude that there is likely an alternative INMT-independent biosynthetic pathway for DMT in mammalian physiology. The neural correlates of intravenous DMT have been investigated in human studies, but so far there have been no studies to investigate the neurochemical or neurophysiological effects of DMT in a rodent model, which is the model of choice for mechanistic investigations. Therefore, we implemented an ultra-high-performance liquid chromatography tandem mass spectrometry (uHPLC-MS/MS) approach coupled with open flow microperfusion for in vivo sampling of several neurochemicals from prefrontal (PFC) and somatosensory (S1BF) cortices along with simultaneous high-density EEG recording before, during and after three doses of intravenous DMT in male and female adult rats. In addition to demonstrating that endogenous DMT is present in PFC and S1BF at levels comparable to other monoamine transmitters, we showed that DMT caused acute increases in serotonin and dopamine in both brain regions in a dose-dependent manner. This suggests that the effects of DMT may be in part modulated by its ability to affect levels of endogenous signaling molecules. Furthermore, we report that DMT caused marked EEG changes, including significant reductions in spectral power and corticocortical coherence within the theta (4-8 Hz) bandwidth, along with simultaneous increases in spectral power and coherence in gamma (65-155 Hz) bands. This work challenges a long-standing hypothesis about mammalian DMT biosynthesis and motivates future studies to identify regulatory mechanisms and functional roles of this putative neurotransmitter. It also represents the most robust characterization of the neurochemical and neurophysiological effects of DMT in rodents to date.