Summary Controlled plant growth requires regulation through a variety of signaling molecules, including steroids, peptides, radicals of oxygen and nitrogen, as well as the ‘classical’ phytohormone groups. Auxin is critical for the control of plant growth and also orchestrates many developmental processes, such as the formation of new roots. It modulates root architecture both slowly, through actions at the transcriptional level and, more rapidly, by mechanisms targeting primarily plasma membrane sensory systems and intracellular signaling pathways. The latter reactions use several second messengers, including Ca2+, nitric oxide (NO) and reactive oxygen species (ROS). Here, we investigated the different roles of two auxins, the major auxin indole‐3‐acetic acid (IAA) and another endogenous auxin indole‐3‐butyric acid (IBA), in the lateral root formation process of Arabidopsis and maize. This was mainly analyzed by different types of fluorescence microscopy and inhibitors of NO production. This study revealed that peroxisomal IBA to IAA conversion is followed by peroxisomal NO, which is important for IBA‐induced lateral root formation. We conclude that peroxisomal NO emerges as a new player in auxin‐induced root organogenesis. In particular, the spatially and temporally coordinated release of NO and IAA from peroxisomes is behind the strong promotion of lateral root formation via IBA.