Genes encoding the myogenic regulating factors MyoD and myogenin and the structural muscle proteins myosin light chain 2 (MyLC2) and myosin heavy chain (MyHC) were isolated from juvenile Atlantic halibut (Hippoglossus hippoglossus L.). The impact of temperature on their temporal and spatial expression during somitogenesis were examined by incubating halibut embryos at 4, 6 and 8 degrees C, and regularly sampling for whole-mount in situ hybridisation and reverse transcription (RT)-PCR. There were no significant effects of temperature on the onset of somitogenesis or number of somites at hatching. The rate of somite formation increased with increasing temperature, and the expression of MyoD, myogenin and MyHC followed the cranial-to-caudal somite formation. Hence, no significant effect of temperature on the spatial and temporal expression of the genes studied was found in relation to somite stage. MyoD, which has subsequently been shown to encode the MyoD2 isoform, displayed a novel bilaterally asymmetric expression pattern only in white muscle precursor cells during early halibut somitogenesis. The expression of myogenin resembled that previously described for other fish species, and preceded the MyHC expression by approximately five somites. Two MyLC2 cDNA sequences were for the first time described for a flatfish, probably representing embryonic (MyLC2a) and larval/juvenile (MyLC2b) isoforms. Factors regulating muscle determination, differentiation and development have so far mostly been studied in vertebrates with external bilateral symmetry. The findings of the present study suggest that more such investigations of flatfish species could provide valuable information on how muscle-regulating mechanisms work in species with different anatomical, physiological and ecological traits.