Restriction enzyme digests of bovine genomic DNA were hybridized against a .37-kilobase (kb) quail embryonic myosin heavy-chain (MHC) copy deoxyribonucleic acid (cDNA) probe; containing both translated and nontranslated regions surrounding the 3' end of the gene. These experiments revealed seven to eight different bands of hybridization, indicative of multiple genes of MHC in the bovine genome. Additionally, a bovine genomic recombinant DNA library was screened with the .37-kb probe. Of the 10(6) phage screened, 11 clones containing portions of the MHC genome were identified, and four were selected for further analyses. Characterization of these four clones was carried out by constructing partial restriction enzyme maps of the inserts using six restriction enzymes singly or in combination. Orientation of the inserts with respect to the arms of the vector and with respect to direction of transcription was determined by hybridizing the DNA fragments against either the .37-kb Pst 1 fragment of pcC128 or to the .23-kb Pst I fragment of pcC128. The .23-kb fragment is located upstream from the .37-kg fragment and contains only coding sequence. Therefore, the differential hybridization pattern of these two probes provided a means for determining the probable direction of transcription. These data provide evidence for a myosin multigene family in cattle, as well as illustrating that the organization of these genes around the 3' end is unique for each of the genes analyzed.