The uncoupling of the calf thymus Topoisomerase I-mediated forward DNA cleavage reaction from the religation event by a rapid shift of cleavage temperature either from 37 degrees C to 0 degrees C or from 37 degrees C to 56 degrees C has been studied and utilized to elucidate the molecular mechanism by which camptothecin, a clinically relevant antineoplastic agent, influences the half reactions of the enzyme. Results of heating and cooling religation-inducing treatments have been compared: both temperature extremes reduce the amount of protein-linked DNA breaks to background levels, thereby affecting cleavage reversal. Camptothecin is found to stabilize the enzyme-DNA intermediate, by inhibition of the Topoisomerase I-mediated rejoining of cleaved DNA, even when the drug is added after formation of the complex. We conclude that: 1. Heating and cooling treatments show a pronounced effect on the DNA cleavage-religation equilibrium. The efficacy of cold is more pronounced than that of heat. 2. Reversal of the enzyme-DNA intermediate favors the DNA resealing versus the closed relaxed form. 3. Camptothecin affects the heat or cold induced religation: in fact in both cases the drug delays the religation step.