Immunoreactivity for calretinin, a calcium-binding protein, was studied in neurones in the guinea-pig small intestine. 26 +/- 1% of myenteric neurones and 12 +/- 3% of submucous neurones were immunoreactive for calretinin. All calretinin-immunoreactive neurones were also immunoreactive for choline acetyltransferase and hence are likely to be cholinergic. In the myenteric plexus, two subtypes of Dogiel type-I calretinin-immunoreactive neurones could be distinguished from their projections and neurochemical coding. Some calretinin-immunoreactive myenteric neurones had short projections to the tertiary plexus, and hence are likely to be cholinergic motor neurones to the longitudinal muscle. Some of these cells were also immunoreactive for substance P. The remaining myenteric neurones, immunoreactive for calretinin, enkephalin, neurofilament protein triplet and substance P, are likely to be orad-projecting, cholinergic interneurones. Calretinin immunoreactivity was also found in cholinergic neurones in the submucosa, which project to the submucosal vasculature and mucosal glands, and which are likely to mediate vasodilation. Thus, calretinin immunoreactivity in the guinea-pig small intestine is confined to three functional classes of cholinergic neurones. It is possible, for the first time, to distinguish these classes of cells from other enteric neurones.