This investigation demonstrates the presence and binding of the protein LC8 (described as "protein inhibitor of nNOS" or PIN in initial reports) to different components of nNOS in nitrergic varicosities of mice gut. Whole varicosity extracts showed three (320, 250 and 155kDa) nNOS bands with anti-nNOS_1422-1433 antibody and a 10kDa band with anti-LC8 antibody. The LC8-immunoprecipitate (IP) showed three nNOS bands, suggesting that LC8 was bound with all three forms of nNOS but dissociated from them during SDS-PAGE. Studies using LC8-immunoprecipitate and supernatant and probed with anti-CaM showed that LC8 was not associated with CaM-bound 320kDa nNOS, but was present in CaM-lacking fraction. Probing these fractions with anti-serine847-P-nNOS showed that 320kDa serine847-phosphorylated-nNOS consisted of LC8-bound and LC8-lacking components. Subsequent studies with varicosity membrane and cytosolic fractions separately showed that membrane contained CaM-bound and CaM-lacking, serine847-phosphorylated 320kDa nNOS; both these fractions lacked LC8. On the other hand, the cytosolic fraction contained CaM-lacking, serine847-phosphorylated 320kDa, 250kDa and 155kDa nNOS bands that were all associated with LC8. These studies, along with in vitro nitric oxide assays, show that in gut nitrergic nerve varicosities: 1) all cytosolic serine847 phosphorylated nNOS was catalytically inactive and bound with LC8. 2) Membrane-associated nNOS consisted of catalytically active, CaM-bound and catalytically inactive, CaM-lacking, serine847-phosphorylated nNOS dimers, both of which lacked LC8. These results suggest that LC8 may dissociate from the 320kDa nNOS dimer upon binding to membrane, thus supporting the view that LC8 may transport nNOS dimer to the varicosity membrane for participation in nitrergic neurotransmission.