We are interested in the study of the molecular basis of human genetic diseases, with an emphasis on inborn errors of metabolism associated with proline and ornithine metabolism. We used a combination of molecular approaches and yeast genetics to clone and characterize 6 genes encoding 5 enzymes in the pathways interconverting glutamate, ornithine and proline. For example, human P5C dehydrogenase (P5CDh) is a mitochondrial matrix enzyme that catalyzes the conversion of P5C, derived either from proline or ornithine, to glutamate. Deficiency of P5CDh causes HPII, an autosomal recessive disorder characterized by seizures and accumulation of proline and P5C. Utilizing peptide sequence data and degenerate primer PCR, we cloned putative full length human P5CDh cDNAs. To confirm the identity of these cDNAs, we transfected them into a P5CDh-deficient S. cerevisiae strain and showed they restored the ability of these cells to grow with proline as a sole nitrogen source and yielded detectable P5CDh activity. Similarly we have expressed human ornithine-δ-aminotransferase (OAT), P5C reductase and P5C synthase in yeast strains carrying mutations of the orthologous genes and shown complementation. We have also used this system to test the functional significance of mutations in OAT and P5CDh. We conclude that these auxotrophic yeast mutants provide a powerful system for cloning, expression and functional analysis of human genes involved in inborn errors of metabolism.