The lateral efferent (olivocochlear) innervation of the cochlea originates in the brainstem lateral superior olive. It is likely to use acetylcholine, γ-aminobutyric acid, dopamine and various neuropeptides as neurotransmitters and/or neuromodulators. In order to determine the different coexistence patterns of these molecules in lateral efferent perikarya, we have used double and triple immunofluorescence co-localization techniques to colocalize choline acetyltransferase, glutamate decarboxylase, tyrosine hydroxylase, calcitonin gene-related peptide and enkephalins in single sections of the lateral superior olive. We also used a non-radoactive in situ hybridization technique onto serial sections of this nucleus to confirm the immunofluorescence co-localization data at the mRNA level. Whatever the pair or triplet of primary antibodies tested was, a high ratio of coexistence was observed in the immunofluorescence experiments. In triple co-localization experiments, 90–93% of the choline acetyltransferase-like immunoreactive neurons were also immunoreactive to the two other antigens investigated. The in situ hybridization co-localization data, based on the use of biotin-labelled oligoprobes, qualitatively confirmed these immunofluorescence data. In conclusion, it can be postulated that acetylcholine, γ-aminobutyric acid, dopamine, calcitonin gene-related peptide, enkephalins and dynorphins (whose coexistence with choline acetyltransferase and enkephalins has been previously described immunocytochemically) coexist in lateral efferent neurons. Based on these results, it is tempting to propose the lateral efferent innervation as a useful model with which the functional implications of the coexistence of neurotransmitters/neuromodulators can be investigated in vivo.