The chromosomal location of the human gene coding for both the β-subunit of prolyl 4-hydroxylase (P4HB) and the enzyme disulfide isomerase (PDI) was determined using mouse × human somatic cell hybrids and three different methods for identifying either the human P4HB/PDI protein or the respective gene: immunoblotting with species-specific monoclonal antibodies; radioimmunoassay with species-specific polyclonal antibodies; and (3) Southern blotting after cleavage of the DNA with EcoRI, HindIII, or BamHI, followed by hybridization with a mixture of two cDNA probes for human P4HB. All three methods gave identical data, demonstrating complete cosegregation of the human protein or its gene in all 17 cell hybrids tested with human chromosome 17. A cell hybrid lacking an intact chromosome 17 localized the gene to 17p11→q