Amino acid analysis of the culture medium was carried out in a human leukemic lymphoblastoid cell line (REH) established from the lymphoblasts of a patient with acute lymphoid leukemia. The results are compared with those of a reference cell line (LHN13) established from normal human lymphocytes. The most striking difference between these two cell lines concerns proline. In LHN13 the concentration of this amino acid in the culture medium increases by 40 microgram/ml/10(6) cells during a 72-hr incubation. In REH there is a decrease under the same culture conditions. In both cell lines proline is derived from glutamic acid and from arginine, as found with the use of 14C-labeled precursors. Synthesis of proline in the REH line represents approximately 26% of the value measured in LHN13 when the precursor is glutamic acid and 15% when the precursor is arginine. The radioisotopic assay for delta1-pyrroline-5-carboxylate reductase showed that there is a deficiency of this enzyme in the REH cells. The defect in proline synthesis of REH was found at the establishment of this line and constitutes a metabolic marker that has persisted for more than 2 years.