Human prolidase (PEPD, iminodipeptidase, EC 3.4.13.9) and related deficiencies were analyzed in terms of the nature and molecular biology of the enzyme and the molecular events seen in patients with this deficiency. The analyses were based on findings concerning isolation of the enzyme, development of specific antibodies and molecular cloning of cDNA and genome DNA of human prolidase. The studies revealed that human prolidase is a homo-dimer of an identical subunit 492 amino acid residues. The gene for prolidase (PEPD gene) was localized on chromosome 19, spanned more than 130 x 10(3) base-pairs and split into 15 exons. Molecular defects in prolidase deficiency were then analyzed. Two patients with the polypeptide-positive phenotype of the disease carried a mis-sense mutation of exon 12. Two siblings with a polypeptide-negative phenotype carried a gene deletion that encompassed exon 14. These mutations were not found in ten other patients with the disease, hence the molecular defects in prolidase deficiency are apparently highly heterogeneous.