Type II hyperprolinemia is an inherited abnormality in amino acid metabolism characterized by elevated plasma proline concentrations, iminoglycinuria, and the urinary excretion of delta1-pyrroline compounds. To define the enzymologic defect of this biochemical disorder, we developed a specific, sensitive radioisotopic assay for the proline degradative enzyme delta1-pyrroline-5-carboxylic acid dehydrogenase. Using this assay, we have shown an absence of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in the cultured fibroblasts from three patients with type II hyperprolinemia. We confirmed this result on cultured cells by demonstrating a similar absence of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in extracts prepared from the peripheral leukocytes of these patients. Additionally, we found significantly decreased levels of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in the leukocyte extracts from five obligate heterozygotes for type II hyperprolinemia. We also demonstrated a reduction in leukocyte delta1-pyrroline-5-carboxylic acid dehydrogenase activity in three successive generations of a family. These results prove that an absence of delta1-pyrroline-5-carboxylic acid dehydrogenase is the enzymologic defect in type II hyperprolinemia and that this defect is inherited in an autosomal recessive fashion.