The binding of [3H]5-hydroxytryptamine (5-HT) to rat enteric membranes was inhibited by the inclusion of 5-HT 2-methyl-5-HT, 5-hydroxytryptophan, N,N,N-triethyltryptamine and 2-Br-N,N-diethyltryptamine in the incubation buffer. In contrast, tryptamine, 5-methoxytryptamine and 2-methyl-N,N-diethyltryptamine enhanced binding. Ascorbate and dithiothreitol facilitated and reduced binding at low and high concentrations respectively. Methysergide, tropisetron, paroxetine and pargyline failed to modify binding. However, following the establishment of [3H]5-HT binding, the subsequent addition of 5-HT and other agents failed to displace [3H]5-HT binding. Heat treatment of the membrane preparation also failed to modify [3H]5-HT binding. The latter findings of an irreversible and heat-insensitive binding indicate that the apparently high affinity 'specific' labelling of [3H]5-HT to rat enteric membranes does not represent receptor-bound [3H]5-HT. The complex interaction between [3H]5-HT and other agents may reflect oxidative events and problematic binding.