The aim of this study was to characterize neuronal nitric oxide synthase (nNOS) activity and 5'-end splice variants in rat small intestine. nNOS was predominantly expressed in the longitudinal muscle layer, with attached myenteric plexus (LM-MP). The biochemical properties of NOS activity in enriched nerve terminals resemble those of nNOS isolated from the brain. Western blot analysis of purified NOS protein with an nNOS antibody showed a single band in the particulate fraction and three bands in the soluble fraction. Rapid amplification of 5' cDNA ends-PCR revealed the presence of three different 5'-end splice variants of nNOS. Two variants encode for nNOSalpha, which has a specific domain for membrane association. The third variant encodes for nNOSbeta, which lacks the domain for membrane association and should therefore be soluble. nNOS is predominantly expressed in LM-MP and can be enriched in enteric nerve terminals. We present the first evidence that three 5'-end splice variants of nNOS encoding two different proteins are expressed in rat small intestine. These two nNOS enzymes exhibit different subcellular locations and might be implicated in different biological functions.