A fragment of a bovine myosin heavy-chain (MHC) gene approximately 15 kbp in size (designated MHC 67) was isolated from a bovine genomic DNA library. The direction of transcription was determined, and preliminary experiments indicated that the gene was expressed in fetal skeletal muscle. The expression pattern of this gene was, therefore, evaluated in detail using northern blots containing RNA from eleven different bovine muscle and nonmuscle tissues at three developmental ages. A restriction fragment of clone MHC 67 containing the 3' untranslated sequence (which is specific for each MHC gene) was used as a probe. This gene fragment hybridized predominantly to RNA from fetal skeletal muscles and did not hybridize to RNA from either neonatal or adult skeletal muscles (red or white), smooth muscle tissue, or nonmuscle tissue. A 7-kb EcoRI fragment containing both translated and untranslated regions surrounding the 3' end of the gene was subcloned into pBluescript II KS+ and partially sequenced. When these bovine sequences were aligned to that of the human and rat skeletal and cardiac MHC genes, we found that these sequences corresponded to exons 31, 32, and 33, and that they had homology with human perinatal and fetal MHC as high as 90% at the nucleotide level and 97% at the amino acid level. Comparison of the nucleotide sequences of isoform-specific 3' nontranslated regions from bovine, human, and rat genes further verify that the MHC 67 clone encodes the bovine fetal or perinatal MHC isoform.