The pattern of distribution and co-localization of nitric oxide synthase (NOS) and quinacrine fluorescence (indicative of vesicular adenosine 5'-triphosphate, ATP), and co-localization of NADPH-diaphorase (NADPH-d) activity and NOS-immunoreactivity in the myenteric plexus of pre-term human fetal (6–17 weeks of gestation) stomach and small intestine was examined using immunohistochemical and histochemical techniques. In all stages of gestation investigated, NOS-immunoreactive and NADPH-d-reactive myenteric neurons and nerve fibres were seen in the fetal intestine and stomach. However, in fetuses of 6–10 weeks of gestation, only 15% of the NADPH-d-positive myenteric neurons were NOS-immunoreactive, whereas a 100% co-localization was found in samples of 12–17 weeks of gestation. Quinacrine fluorescent myenteric neurons and nerve fibres were found only in the fetal intestine of 12–17 weeks of gestation, of which 25% of the NADPH-d-positive myenteric neurons in these samples were quinacrine fluorescent. These findings demonstrate the presence and co-localization of markers for nitric oxide (NO)- and ATP-utilizing myenteric neurons and nerve fibres in the early stages of gestation, suggesting possible co-transmitter and/or trophic roles of ATP and NO in the process of development and maturity of human myenteric neurons. In addition, the fact that only a small percentage of NADPH-d-reactive myenteric neurons express NOS immunoreactivity at 6–10 weeks of gestation confirms that NADPH-d-reactivity does not always represent NOS activity.