BACKGROUND: While combination of gemcitabine with anti-topoisomerase poisons is routinely used in oncology, little is known on the biological interactions between these drugs. DESIGN: To understand the cellular basis for this association, we hypothesized an interaction of the two agents at the topoisomerase level. A real-time RT-PCR method was designed to quantify topoisomerase expression after treatment with gemcitabine (GEM) in two human colon adenocarcinoma cell lines. Efficacy of drugs as single agents and in combination was analyzed on the basis of their cytotoxic effects. RESULTS: We showed that a) gemcitabine induces expression of all major eukaryotic topoisomerases (I, II alpha and beta) at definite times after drug administration; b) cytotoxicity was more relevant when cells were treated with GEM and the topoisomerase poison within a short period of time. In particular synergistic effects were found when the anti-topoisomerase II agent was given 3 h after gemcitabine or when the anti-topoisomerase I drug was delivered 3 h before or after the antimetabolite. CONCLUSIONS: These findings help explaining the effectiveness of the combined therapy GEM/topoisomerase poisons and suggest a drug administration protocol for clinical treatment.