Aminopterin, a folate antagonist, added to cultures of mouse spleen cells stimulated with T-cell mitogens markedly inhibited, in a dose-dependent manner, [3H]thymidine incorporation into the cells. Contrary to this inhibitory effect aminopterin added at the same concentrations to cell cultures stimulated with B-cell mitogens significantly increased [3H]thymidine incorporation. The inhibitory effect of aminopterin on T-cell activation was completely reversed by adding hypoxanthine to the cultures. Analysis of the mechanism of aminopterin action showed direct, mutually opposite, effects of this drug on T- and B-cell populations. The results thus not only demonstrate that polyclonally activated T and B lymphocytes differ in their sensitivity to the toxic action of aminopterin, but simultaneously support early findings suggesting differences in nucleic acid metabolic pathways between T- and B-cell populations.