Proline independence in CHO-K1 Chinese hamster cells has in previous studies been characterized as an auxotrophic gene mutation. In the absence of direct proof, an alternative model must be considered, based on suppression of proline synthesis by DNA methylation changes at one or more loci concerned. This concept receives strong support from the present study, in which we show that treatment of CHO-K1 cells with 5-azacytidine induces a 10(5)-10(6) increase in background conversion to the proline-independent state. Revertants thus obtained, as well as those arising spontaneously or after treatment with ethyl methane sulfonate, are stable phenotypically in the presence or absence of proline. Proline independence in all variants examined was correlated with increased activity of pyrroline-5-carboxylate synthase. Four of five variants induced with 5-azacytidine showed simultaneous increases in activity of ornithine aminotransferase as well. Our data suggest that epigenetic, rather than genetic changes, underlie the transitions between proline dependence and independence in CHO-K1 cells.