The major histocompatibility complex and prolactin (PRL) genes are syntenic in humans and cattle but the genetic distance between these loci has not been determined for either species. In this study, the sperm typing technique was used to measure the recombination frequency between the bovine lymphocyte antigen (BoLA)-DRB3 and PRL loci. A total of 300 sperm were typed from one doubly heterozygous bull for segregation of DRB3 and PRL alleles. Sperm typing was performed using the polymerase chain reaction (PCR) and restriction enzyme cleavage of the PCR products, followed by resolution of the restriction fragments in polyacrylamide gels. Digestion with the restriction endonuclease RsaI allowed the unambiguous discrimination of alleles for both loci. The maximum likelihood estimation of the recombination fraction theta = 0.04, with a 95% confidence interval of 0.01 to 0.07. Close linkage between PRL and DRB3 has important implications for marker-assisted selection in animal breeding since PRL has been shown to be closely linked to a locus that affects milk yield, and BoLA loci influence susceptibility to a number of infectious diseases. Our results demonstrate the general applicability of the sperm typing procedure for gene mapping in species other than humans and provide an example of how parallel efforts to map the genomes of agriculturally important species of animals can have a positive impact on the development of a primary human linkage map.