ABSTRACT The fast skeletal muscle myosin light chain 2 (MLC2) gene is expressed specifically in skeletal muscles of new born and adult mice, and has no detectable sequence homology with any of the other MLC genes including the slow cardiac MLC2 gene. The expression of the fast skeletal muscle MLC2 gene during early mouse embryogenesis was studied by in situ hybridization. Serial sections of embryos from 8.5 to 12.5 days post coitum (d.p.c.) were hybridized to MLC2 cRNA and to probes for the myogenic regulatory genes MyoD1 and myogenin. The results revealed different temporal and spatial patterns of hybridization for different muscle groups. MLC2 transcripts were first detected 9.5 d.p.c. in the myotomal regions of rostral somites, already expressing myogenin. Surprisingly, at the same stage, a weak MLC2 signal was also detected in the cardiomyocytes. The cardiac expression was transient and could not be detected at later stages while the myotomal signal persisted and spread to the more caudal somites, very similar to the expression of myogenin. Beginning from 10.5 d.p.c., several extramyotomal premuscle cells masses have been demarcated by MyoD1 expression. MLC2 transcripts were detected in only one of these cell masses. Although, transcripts of myogenin were detected in all these cell masses, the number of expressing cells was significantly lower than that observed for MyoD1. By 11.5 d.p.c., all three hybridization signals colocalized in most extramyotomal muscle forming regions, with the exception of the diaphragm and the hindlimb buds, where only few cells expressed MLC2 and more cells expressed MyoD1 than myogenin. At 12.5 d.p.c., all three studied genes displayed a similar spatial pattern of expression in most muscle-forming regions. However, in some muscles, the MyoD1 signal spread over more cells compared to myogenin or MLC2. Our results are consistent with the suggestion that multiple myogenic programs exist for myoblasts differentiating in the myotome and extramyotomal regions.