Nitric oxide (NO) is responsible for nitrergic neurotransmission in the gut, and its release is dependent on its de novo synthesis by neuronal nitric oxide synthase (nNOS). The magnitude of NO synthesis and release during neurotransmission may be related to the fraction of catalytically active nNOS out of a larger pool of inactive nNOS in the nerve terminals. The purpose of the present study was to identify catalytically active and inactive pools of nNOS in the varicosities from mouse gut. Enteric varicosities were confirmed as nitrergic by colocalization of nNOS with the nerve varicosity marker synaptophysin. Low-temperature SDS-PAGE of these varicosity extracts showed 320-, 250-, and 155-kDa bands when blotted with anti-nNOS(1422-1433) and 320- and 155-kDa bands when blotted with anti-nNOS(1-20) antibodies, respectively. The 320- and 155-kDa bands represent dimers and monomers of nNOSalpha; the 250- and 135-kDa bands represent dimers and monomers of nNOSbeta. Immunoprecipitation with calmodulin (CaM) showed that a portion of nNOSalpha dimer was bound with CaM. On the other hand, a portion of nNOSalpha dimer, nNOSbeta dimer, and all monomers lacked CaM binding. The CaM-lacking nNOS fractions reacted with anti-serine 847-phospho-nNOS. In vitro assays of NO production revealed that only the CaM-bound dimeric nNOSalpha was catalytically active; all other forms were inactive. We suggest that the amount of catalytically active nNOSalpha dimers may be regulated by serine 847 phosphorylation and equilibrium between dimers and monomers of nNOSalpha.