ABSTRACT MRF4 (herculin/Myf-6) is one of the four member MyoD family of transcription factors identified by their ability to enforce skeletal muscle differentiation upon a wide variety of nonmuscle cell types. In this study the mouse germline MRF4 gene was disrupted by targeted recombination. Animals homozygous for the MRF4bh1 allele, a deletion of the functionally essential bHLH domain, displayed defective axial myogenesis and rib pattern formation, and they died at birth. Differences in somitogenesis between homozygous MRF4bh1 embryos and their wild-type littermates provided evidence for three distinct myogenic regulatory programs (My1-My3) in the somite, which correlate temporally and spatially with three waves of cellular recruitment to the expanding myotome. The first program (My1), marked initially by Myf-5 expression and followed by myogenin, began on schedule in the MRF4bh1/bh1 embryos at day 8 post coitum (E8). A second program (My2) was highly deficient in homozygous mutant MRF4 embryos, and normal expansion of the myotome failed. Moreover, expression of downstream muscle-specific genes, including FGF-6, which is a candidate regulator of inductive interactions, did not occur normally. The onset of MyoD expression around E10.5 in wild-type embryos marks a third myotomal program (My3), the execution of which was somewhat delayed in MRF4 mutant embryos but ultimately led to extensive myogenesis in the trunk. By E15 it appeared to have largely compensated for the defective My2 program in MRF4 mutants. Homozygous MRF4bh1 animals also showed improper rib pattern formation perhaps due to the absence of signals from cells expressing the My2 program. Finally, a later and relatively mild phenotype was detected in intercostal muscles of newborn animals.