In vitro folate deficiency is associated with S phase accumulation and apoptosis in various cell types. To investigate the role of p53 and two apoptosis-related molecules, bcl-2 and Fas antigen (Apo-1, CD95), in the mechanism whereby folate-deficient lymphocytes accumulate and undergo apoptosis in the S phase, normal human peripheral blood lymphocytes were cultured for 3–9 d in control medium or in specially ordered and formulated HAM's F-10 medium lacking folic acid, thymidine and hypoxanthine. Cells were stimulated with phytohaemagglutinin for the final 72 h prior to harvesting. The results indicate that p53 expression was downregulated in folate-deficient lymphocytes when compared with the control lymphocytes during the relevant period of S phase accumulation and apoptosis. In addition, folate deficiency was also found to downregulate IL-2, Fas antigen and bcl-2 expression, in terms of either mRNA or protein levels. The downregulation of Fas antigen suggests that folate deficiency-induced apoptosis probably does not occur via the Fas pathway. As IL-2 is a known inducer of bcl-2, and the downregulation of bcl-2 induces apoptosis, the downregulation of IL-2 and bcl-2 is suggested to play an important role in apoptosis. The complete rescue of folate-deficient lymphocytes from apoptosis was achieved by folic acid, thymidine or hypoxanthine alone or thymidine and hypoxanthine in combination. These results suggest that IL-2 depletion by folate deficiency in lymphocytes reduces the bcl-2 level, thereby triggering deoxynucleoside triphosphate pool imbalance and p53-independent apoptosis.