Thymidylate synthase (TS) (EC 2.1.1.45) provides precursors for DNA biosynthesis through a de novo pathway and is a key target enzyme for cancer chemotherapy. TS levels of human leukemic leukocytes from patients with chronic myelocytic leukemia (CML) and acute lymphocytic leukemia (ALL) were observed to be highly elevated (66- and 33-fold for CML and ALL, respectively) compared to the usual low level of basal activity in normal healthy controls. In vitro inhibition studies on the human leukemic leukocyte TS with the phenanthroindolizidine alkaloids pergularinine (PGL) and tylophorinidine (TPD) (isolated from the Indian medicinal herb Pergularia pallida) were conducted for the preliminary screening tests for their antitumor activity. The leukemic leukocyte enzyme activity was potently inhibited by PGL and TPD (IC50 = 50 microM) in both types of leukemias. These alkaloids were assessed for biological evaluation for the first time as potential antileukemic agents.