The mitochondria of rat intestinal mucosa were found to have an enzymatic activity that converts radioactive glutamate to pyrroline-5-carboxylate (P5C) in the presence of ATP, NADPH, and MgCl2. The product of this enzyme was identified as P5C by the fact that it was converted to proline by chemical reduction with NaBH4 or by enzymatic reduction with NADH in the presence of purified yeast P5C reductase. The product was demonstrated to be P5C rather than pyrroline-2-carboxylate by thin layer chromatography. The presence of the activity in mitochondria prepared from intestinal mucosa of germ-free rats proved that this activity is of mammalian origin. Omission of either ATP, NADPH, or MgCl2 from the reaction mixture resulted in little or no activity. The optimal pH appeared to be about 7.0 under the conditions used. Substrate saturation curves in the presence of an ATP and an NADPH regeneration system gave apparent Km values of 2.5 mM for glutamate, 0.19 mM for ATP, and 6.5 microM for NADPH in the presence of 20 mM MgCl2. The mitochondrial preparation usually produced P5C at a rate of 1.2 to 1.6 nmol/mg/min at 20 degrees C when incubated with 1 mM glutamate, 3 mM ATP, 0.2 mM NADPH, and 20 mM MgCl2.