The genes for three proteins of the pyruvate dehydrogenase (PDH) complex have been assigned to human chromosomes by Southern analysis of a panel of human-rodent somatic cell hybrid DNAs with cDNA probes for these genes. PDH-E1 alpha has been localized on human chromosome 3p13-q23. The assignments of lipoamide dehydrogenase(E3) and PDH-E1 alpha [corrected] to chromosomes 7 and Xp, respectively, have been confirmed. Restrictive-fragment-length polymorphisms have been identified with E3, which will permit further localization of this gene by genetic linkage analysis.