Nitric oxide synthase (NOS) immunoreactivity occurs in two groups of neurons in the guinea pig small intestine: descending interneurons that are also immunoreactive for choline acetyltransferase (ChAT), and inhibitory motor neurons that lack ChAT immunoreactivity. Interneurons that are involved in local reflexes would be expected to have inputs from intrinsic primary afferent (sensory) neurons, most of which are calbindin-immunoreactive. We examined this possibility using triple staining for NOS, ChAT and calbindin immunoreactivity and investigated the relationships between calbindin-immunoreactive varicosities and the cell bodies of NOS-immunoreactive neurons, using high-resolution confocal microscopy and electron microscopy. By confocal microscopy, we found that the cell bodies of ChAT/NOS interneurons received 84 +/- 23 (mean +/- SD) direct appositions from calbindin-immunoreactive varicosities and that the cell bodies of NOS-inhibitory motor neurons received 82 +/- 20 appositions. Electron-microscopic examination of the relations of 265-calbindin-immunoreactive varicosities, at distances within the resolution of the confocal microscope (300 nm), to 30 NOS-immunoreactive nerve cells indicated that 84% formed close contacts or synapses and 16% were separated from neurons by thin glial cell processes. Thus, each NOS-immunoreactive nerve cell receives about 70 synaptic inputs or close contacts from the calbindin-immunoreactive varicosities of intrinsic primary afferent neurons. It is concluded that there are monosynaptic reflex connections in which intrinsic primary afferent neurons synapse directly with motor neurons and di- or poly-synaptic reflexes in which ChAT- and NOS-immunoreactive neurons are interneurons, interposed between intrinsic primary afferent neurons and NOS-inhibitory neurons.