The hypothesis was tested that expression of bcl-2 could provide protection against apoptosis induced by cytotoxic drugs via a mechanism which was different from the classical determinants of drug resistance. Sensitivity and resistance to inhibitors of thymidylate synthase (EC 2.1.1. 45) were chosen for study since these drugs have a well-defined and quantifiable locus of action with similarly well defined biochemical sequelae resulting from enzyme inhibition. Human lymphoma cells transfected with the vector alone readily underwent apoptosis after a 36-h exposure to various drugs. For example, 5-fluorodeoxyuridine (0.1 microM) induced 67% apoptosis in vector control cells 24 h after removal of the drug. In contrast, cells treated under identical conditions, but which expressed the bcl-2 protein, showed only basal levels of apoptosis (8%), with no significant fall in viability. Similar results were obtained using two quinazoline-based inhibitors of thymidylate synthase, N10-propargyl-5,8-dideazafolic acid (CB3717) and ICI M247496. Determinants of resistance to these three drugs were investigated. Analysis of the cell cycle, thymidylate synthase levels, and activity showed these to be unchanged by expression of bcl-2. Addition of the drugs brought about equivalent inhibition of proliferation in the presence or absence of bcl-2 expression. 5-Fluorodeoxyuridine treatment reduced TTP synthesis, induced strand breaks in nascent DNA, measured by alkaline elution, and increased the synthesis of thymidylate synthase; these changes preceded the onset of apoptosis and were identical in the vector controls and bcl-2 transfectants. Resistance to thymidylate stress in bcl-2-expressing cells therefore occurred by a mechanism different from those which classically define resistance to this type of cytotoxic drug.