Double staining immunofluorescence techniques were applied to investigate the co-existence of enkephalin, dynorphin and vasoactive intestinal peptide immunoreactivity in the guinea-pig enteric nervous system. Segments of guinea-pig small intestine were incubated for 24h in a culture medium with colchicine. Several subpopulations with overlapping immunoreactivities were found, i.e. neurons with enkephalin and vasoactive intestinal peptide, with enkephalin and dynorphin and with all three neuropeptides. It is probable that subpopulations with dynorphin and vasoactive intestinal peptide and with each neuropeptide only are also present. These subpopulations of enteric neurons with multiple neurochemical subcoding are likely to have different connections and functions.