pH-mediated conversions in the structure of the topoisomerase (topo) I inhibitors camptothecin (CPT) and its analogues have strong implications for the pharmacokinetics and pharmacodynamics of these novel anticancer agents. Because the cell-penetrating and biologically active lactone isomers predominate at acidic conditions, we have tested if low pH potentiates the cytotoxic and antitumor effects of CPT and its water-soluble derivative topotecan (TPT). In L1210 leukemia cells, rapid initial uptake of radiolabeled CPT and TPT was followed by a gradual release from cells at physiological pH 7.4, whereas high drug levels were maintained in cells at pH 6.2. Steady-state uptake levels of CPT increased proportionally, up to 5-fold, with decreasing pH of the incubating medium (from 7.4 to 6.0). With TPT, a maximum 3-fold increase was observed at pH 6.8 to 6.4. By contrast, the cellular pharmacokinetics of the topoisomerase II inhibitor etoposide (ETP) were independent of the ambient pH. The large increases in intracellular CPT and TPT levels caused only moderate potentiation of cytotoxicity in short-term incubations. Conditions of very low pH < or =6.2 even antagonized the cytotoxicity of the topo I and topo II inhibitors, due to inhibition of DNA synthesis by intracellular acidification. However, in clinically relevant schedules of prolonged exposures at low drug concentration, low pH potentiated the cytotoxicity of CPT and TPT by 2-3-fold. To investigate the effect of local pH in vivo, the basal interstitial pH of 6.8 of RIF-1 tumors was selectively lowered by i.p. injection of the host animals with the mitochondrial inhibitor meta-iodobenzylguanidine (32 mg/kg) and glucose (1.5 g/kg). In accordance with the pH optimum for TPT uptake at pH 6.8 to 6.4, tumor acidification had no effect on the antitumor effect of this analogue. By contrast, the intervention significantly potentiated the response of tumors to CPT. The results indicate that local pH is an important determinant of the cellular pharmacokinetics and the antitumor activity of CPT and analogues.