To identify brain structures potentially acting as biological clocks in rainbow trout (Oncorhynchus mykiss), the expression sites of a trout homolog of the mouse clock gene were studied and compared with that of melatonin receptors (Mel-R). For this purpose, a partial sequence of the trout clock gene, including a PAS domain, was obtained by reverse transcription-polymerase chain reaction and used to perform in situ hybridization. The highest density of clock transcripts was observed in the periventricular layer (SPV) of the optic tectum, but a weaker expression was detected in some pretectal nuclei, such as the posterior pretectal nucleus (PO) and the periventricular regions of the diencephalon. Comparison of the hybridization signal in fish sacrificed at 08:00 and 17:00 did not indicate major changes in clock expression levels. Comparison of adjacent sections alternatively treated with clock and Mel-R probes suggests that both messengers are probably expressed in the same cells in the SPV and PO. In addition, in situ hybridization with a glutamate decarboxylase 65 probe, demonstrates that cells expressing clock and Mel-R in the optic tectum are gamma-aminobutyric acid neurons. The tight overlapping between the expression of Mel-R and clock transcripts in cells of the PO and SPV suggests a functional link between these two factors. These results indicate that the optic tectum and the pretectal area of the rainbow trout are major sites of integration of the melatonin signal, express the clock gene, and may act as biological clocks to influence behavioral and endocrine responses in trout.