The effects of CPT on the calf thymus Topoisomerase I-mediated DNA breakage-reunion reaction were studied at an enzyme concentration range proper for evidencing, at the same time, both DNA relaxation and DNA cleavage/religation. Some of the requirements and the optimal conditions for the formation and reversal of the CPT-trapped Topoisomerase I-DNA cleavable complex are also characterized. We conclude that: 1. Calf thymus (100 kDa) Topoisomerase I requires, for maximal DNA cleavage activity, specific and characteristic reaction conditions. 2. CPT does not affect these optimal conditions, but only stabilizes the normal enzyme-DNA intermediate. In this way, the drug lowers the religation process, becoming responsible for the relaxation inhibition. 3. The optimum of monovalent salt concentration for cleavable complex formation is found between 30 and 70 mM. These values are lower than those required for the relaxation activity optimum (75-125 mM NaCl). 4. The addition of 0.5 M monovalent salt causes reversal of the reaction, and shifts the equilibrium distribution between cleavable intermediate and closed relaxed DNA in the direction of DNA resealing. Therefore, it is suggested that salt affects the cleavage but not the religation reaction.