The 5-HT(3) receptor is a member of the superfamily of neurotransmitter-gated ion channels involved in fast synaptic signalling and in modulation of neurotransmitter release. As for many other channel receptors, the electrophysiological properties and the functions of the 5-HT(3) receptor are determined by subunit composition of the pentameric channel. Because in situ hybridization did not allow the detection of mRNA encoding the 5-HT(3B) subunit in the rodent central nervous system, or in nearly half of the neurons expressing the 5-HT(3A) subunit in peripheral ganglia, it has been suggested that subunit composition could define at least two 5-HT(3) receptor-expressing neuronal populations. In order to challenge this hypothesis, we have developed polyclonal antibodies directed against a portion of the second intracytoplasmic loop of the mouse 5-HT(3B) subunit. Immunohistochemical analysis in the mouse and the rat revealed that immunolabelling was most prominent in peripheral ganglia, particularly in trigeminal ganglia (TG). In rats, transection or ligature of the infraorbital nerve resulted in a pronounced accumulation of immunoreactive material at the proximal side of the lesioned nerve, and an up-regulation of both subunits in 5-HT(3) receptor-expressing TG neurons. Surprisingly, nearly 100% of neurons expressing 5-HT(3A) subunits were also labelled by anti-5-HT(3B) antibodies. We also detected 5-HT(3B) immunoreactivity in the rat hippocampal CA1 layer and in scattered cortical neurons, indicating that detection of 5-HT(3) subunit mRNA by in situ hybridization might not provide really complete mapping of heteromeric 5-HT(3A/B) vs. homomeric 5-HT(3A) receptors in the peripheral and central nervous systems in rodents.