Retroviral integrases catalyze four endonuclease reactions (processing, joining, disintegration, and nonspecific alcoholysis) that differ in specificity for the attacking nucleophile and target DNA sites. To assess how the two substrates of this enzyme affect each other, we performed quantitative analyses, in three retroviral systems, of the two reactions that use a variety of nucleophiles. The integrase proteins of human immuno- deficiency virus type 1, visna virus, and Rous sarcoma virus exhibited distinct preferences for water or other nucleophiles during site-specific processing of viral DNA and during nonspecific alcoholysis of nonviral DNA. Although exogenous alcohols competed with water as the nucleophile for processing, the alcohols stimulated nicking of nonviral DNA. Moreover, different nucleophiles were preferred when the various integrases acted on different DNA targets. In contrast, the nicking patterns were independent of whether integrase was catalyzing hydrolysis or alcoholysis and were not influenced by the particular exogenous alcohol. Thus, although the target DNA influenced the choice of nucleophile, the nucleophile did not affect the choice of target sites. These results indicate that interaction with target DNA is the critical step before catalysis and suggest that integrase does not reach an active conformation until target DNA has bound to the enzyme.