Staphylococcus aureus is one of the most common causes of bovine mastitis. To estimate genetic relationships among S. aureus strains recovered from cows, 357 isolates from milk samples from worldwide localities were examined for electrophoretic variation at 13 metabolic-enzyme loci. Thirty-nine electrophoretic types which represented distinctive multilocus enzyme genotypes were identified, and nearly 90% of all isolates were assigned to one of eight clones. Genetic heterogeneity was found among organisms recovered from dairy herds from which multiple isolates were obtained, indicating that the S. aureus population in a single herd can be multiclonal. Although humans and cows shared 7 of the 39 S. aureus clones, each clone was predominantly associated with one of these host species. These results are consistent with the concept of host specialization among S. aureus clones and imply that successful transfer of bacteria between humans and cows is limited.