Cyclic 3',5'-adenosine monophosphate (cAMP) is a critical and ubiquitous second messenger involved in a multitude of signaling pathways. Soluble adenylyl cyclase (sAC) is a novel source of cAMP subject to unique localization and regulation. It was originally discovered in mammalian testis and found to be activated by bicarbonate and calcium. sAC has been implicated in diverse processes, including astrocyte-neuron metabolic coupling and axonal outgrowth of neurons. However, despite these functional studies, demonstration of sAC protein expression outside of testis has been controversial. Recently, we showed sAC immunoreactivity in astrocytes, but the question of neuronal expression of sAC remained. We now describe the generation of a second sAC knockout mouse model (C2KO) designed to more definitively address questions of sAC expression, and we demonstrate conclusively using immune-electron microscopy that sAC is expressed in neuronal profiles in the central nervous system.