Purpose: The influence of biophysical factors (drug metabolism, transport proteins, and chemical stability) on the cellular accumulation of camptothecin (CPT) and SN-38 was examined. Methods: Drug transporter RNA transcript levels were measured by real-time reverse transcriptase polymerase chain reaction (RTPCR). Intracellular drug accumulation, metabolism, and drug stability studies were all performed by HPLC. Results: A panel of three human cell lines exhibiting different drug resistant phenotypes was investigated. HT29 colon cells glucuronidated SN-38 but did not express P-gp or MRP1 or 2. HCT116 colon cells expressed P-gp and MRP2 but did not catalyse conjugation. A2780 ovarian cells neither catalysed drug metabolism nor contained these drug transporters. In all lines, SN-38 lactone was rapidly taken up achieving peak concentrations at the earliest time point studied (5 min, 3.3-4.1 ng/10(6) cells). Subsequently, a fall in intracellular lactone concentration occurred, stabilising after 4 h at 0.48-1.18 ng/10(6) cells. No significant differences in intracellular levels of lactone were observed between the three cell lines with one exception: a twofold increase in HCT116 cells at 24 h. Stability studies in culture medium revealed that SN-38 lactone concentrations disappeared at the same rate regardless of whether cells were present, initially falling to reach equilibrium with the hydroxy acid by 4 h. Indeed, changes in intracellular lactone concentrations followed closely chemical stability profiles in media. Similar patterns of cellular retention and chemical degradation were observed with CPT. Conclusion: The major determinant of drug accumulation in three diverse cell line phenotypes was lactone chemical stability in culture medium.