SummaryA swine genomic cosmid library constructed from a genotypically SLA homozygous Large White individual was screened with a murine genomic 21‐hydroxylase probe. A clone which contained a pig 21‐hydroxylase gene was isolated and after subcloning, the 5′ region of the gene was sequenced. The deduced amino acid sequence corresponded almost exactly to the NH2terminal portion of the steroid 21‐hydroxylase from porcine adrenal microsomes. Comparison of the first 99 amino acid residues of both sequences revealed three substitutions comprising two leucine residues in positions 10 and 13, and one arginine residue in position 55 for our sequence, instead of threonine in position 10 and lysine in position 13 and 55 for the isolated enzyme. A swine homologous probe was derived from the isolated 21‐hydroxylase gene and used for gene assignment by RFLP studies in two swine leucocyte antigen (SLA) informative families. The results demonstrate that the swine 21‐hydroxylase gene is located within or close to the swine MHC. Taken together, the present results suggest the existence of a single 21‐hydroxylase gene per h