Mutants of Chinese hamster ovary cells (CHO-K1 Pro-), resistant to the proline transport antagonist 2-(methylamino)-isobutyrate (MeAIB) were isolated by a single-step procedure. Mutation rates to Pro+ and to Pro- MeAIB resistance (MeAIBr) are 1.7 X 10(-6) and 2.4 X 10(-5), respectively. Several Pro- MeAIBr mutants were tested by measuring the uptake of 0.05 mM proline through the various amino acid transport systems: some showed increases in one transport system only; others revealed pleiotropic changes affecting two or more systems; still others had no apparent change in proline transport. One Pro- MeAIBr mutant analyzed in detail (MeAIBr22) was isolated after EMS treatment as resistant to 5 mM MeAIB, is Pro-, stable, and shows a 1.6-fold increase in the initial velocity of transport of 0.05 mM proline. There appears to be no change in the velocity of proline transport through the amino acid transport systems A, P, and L, and the "glutamine inhibitable fraction." In contrast, there is a 5.5-fold increase in the velocity of transport of 0.05 mM proline through the ASC system. Kinetic studies reveal a sixfold increase in the Vm and a slight increase in the Km of the transport of serine through the ASC system. Hybrids between MeAIBr22 and CHO-K1 Pro-, OUAr, HPRT- showed the parental phenotype. These results indicate that the mutant ASC phenotype of MeAIBr22 is recessive and is probably the result of a regulatory gene mutation.