Gemcitabine (2',2'-difluorodeoxycytidine) is an antineoplastic agent with clinical activity against ovarian carcinoma, small cell and non-small cell lung cancers, head and neck cancer, bladder cancer, breast cancer, and pancreatic cancer. Cisplatin (CDDP), etoposide (VP-16), and mitomycin C (MMC) are well-known anticancer agents that are also active against many of these types of cancer. Because of the low toxicity profile of gemcitabine and the differences in mechanism of cytotoxicity, combinations of these drugs with gemcitabine were studied in vitro and in vivo. Cells were exposed in vitro for 1, 4, 24, or 72 hours to gemcitabine in combination with these drugs, either simultaneously or sequentially in a constant ratio. Another approach consisted of exposure to a combination of the approximate IC25 of one drug and varying concentrations of the other drug. Synergism for several of these combinations was found in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its gemcitabine-resistant variant AG6000, and in the non-small cell lung cancer cell lines H322 and Lewis lung (LL) after a 72-hour drug treatment. Studies of the possible mechanisms of action initially focused on the major metabolic features of each drug. CDDP did not enhance the accumulation of gemcitabine triphosphate and caused only marginal changes in the extent of DNA double-strand breaks (DSBs) induced by gemcitabine in these cell lines. Gemcitabine increased platinum accumulation only in the ADDP cell line, but the DNA platination was enhanced in the A2780, ADDP, AG6000, and LL cell lines. MMC did not influence the formation of DSBs by gemcitabine in the LL cell line. The combination of VP-16 and gemcitabine, however, resulted in the formation of more DSBs in this cell line than each drug alone. This effect was even more pronounced when cells were exposed to VP-16 4 hours before gemcitabine. In vivo, the antitumor activity of a combination of 50 mg/kg gemcitabine and 6 mg/kg CDDP was more effective against LL tumors than each compound alone. In conclusion, gemcitabine is an attractive drug to combine with a wide range of anticancer drugs; synergism is often schedule dependent.