Nitric oxide has been implicated as a local modulator of several gastrointestinal functions. In this study, we have measured nitric oxide synthase activity in homogenates of enterocytes isolated from post-weaned pigs. The enzyme required the presence of NADPH and 6-(R,S)-5,6,7,8-tetrahydro-L-biopterin. Conversely exogenous FAD and FMN did not appear to be necessary for enzyme activity. The enzyme activity was not affected by added Ca2+ or EGTA and was inhibited by the arginine analogs NG-monomethyl-L-arginine and N omega-nitro-L-arginine. NO synthase activity was not detectable in enterocytes isolated at birth and increased slightly in suckling animals. NO synthase activity was found to be present mostly in the cytosolic fraction isolated from post-weaned pigs enterocytes.