It has previously been shown that the antiprotozoal drug pyrimethamine (PYR) in concentrations corresponding to those obtained in clinical practice temporarily suppressed the proliferation of phytohaemagglutinin (PHA-) stimulated human lymphocytes in vitro; 10-fold higher concentrations permanently suppressed PHA-stimulated cells, as indicated by decreased numbers of cells and DNA synthesis. In the present study, it was found that the 3H-deoxyuridine incorporation in PHA-stimulated lymphocytes was suppressed by PYR, and that PYR caused defective deoxyuridine suppression of 14C-thymidine incorporation. The effects of PYR were completely corrected by low concentrations of folinic acid and high concentrations of folic acid, indicating that the basic mechanism of action of PYR is competitive blocking of dihydrofolate reductase. However, the effect of PYR was poorly corrected by exogenous thymidine; therefore, reduced thymidylate synthesis cannot be the sole consequence of PYR exposure. It is suggested that an additional folate-dependent factor plays an important role in the antimitotic activity of PYR on lymphocytes.