Although glutamic acid is known to be a precursor for proline biosynthesis, the enzymatic conversion of glutamic acid to pyrroline-5-carboxylic acid, the immediate precursor of proline, has not been demonstrated in cell-free systems. By providing appropriate concentrations of ATP and NADPH and blocking further metabolism of pyrroline-5-carboxylic acid, we have developed a method for measuring the formation of pyrroline-5-carboxylic acid from glutamic acid in homogenates of mammalian cells. We have designated this activity pyrroline-5-carboxylate synthase. To confirm that our assay is a valid measure of the initial step in proline biosynthesis from glutamic acid, we have compared two mutant lines of Chinese hamster ovary cell. Proline prototrophic cells, which can synthesize proline from glutamic acid, have easily measurable pyrroline-5-carboxylate synthase activity (5.97 nmol of pyrroline-5-carboxylic acid per hr per mg of homogenate protein). In contrast, proline auxotrophic cells, which are unable to synthesize proline from glutamic acid, have no detectable pyrroline-5-carboxylate synthase activity.