Biological activity of the phenanthroindolizidine alkaloids pergularinine (PGL) and tylophorinidine (TPD) isolated from the Indian medicinal herb Pergularia pallida has been evaluated and assessed for the first time employing thymidylate synthase (TS) (5,10-CH2H4 PteGlu: dUMP-C-methyltransferase, EC 2.1.1.45), a key target enzyme in cancer chemotherapy. TS used in the present investigations was purified from Lactobacillus leichmannii. Toxicity studies showed that PGL and TPD were potently toxic and inhibited growth of L.leichmannii cells. Both PGL and TPD significantly inhibited TS activity (IC50 = 40 and 45 microM, respectively). PGL concentrations > 80 microM and TPD concentrations > 90 microM resulted in a complete loss of the TS activity, thus suggesting that both these phenanthroindolizidine alkaloids are promising potential antitumor agents. Our results show that the alkaloid-binding to TS is irreversibly tight through a probable covalent linkage. Inhibition kinetics reveal that the enzyme has Ki values of 10 x 10(-6) and 9 x 10(-6) M for PGL and TPD, respectively and that the inhibition in both the cases is a simple linear 'noncompetitive' type.