The site-specific recombinase Intl1, encoded by class 1 integrons, catalyses the integration and excision of gene cassettes by recognizing two classes of sites, the integron-associated attl1 site and the 59-base element (59-be) family of sites that are associated with gene cassettes. Intl1 includes the four conserved amino acids that are characteristic of members of the integrase family, and Intl1 proteins with single amino acid substitutions at each of these positions had substantially reduced catalytic activity, consistent with this classification. Intl1 was purified as a fusion protein and shown to bind to isolated attl1 or 59-be recombination sites. Binding to attl1 was considerably stronger than to a 59-be. Binding adjacent to the recombination cross-over point was not detected. A strong Intl1 binding site within attl1 was localized by both deletion and footprinting analysis to a 14 bp region 24-37 bp to the left of the recombination cross-over point, and this region is known to be critical for recombination In vivo (Recchia et al., 1994). An imperfect (13/15) direct repeat of this region, located 41-55 bp to the left of the recombination cross-over point, contains a weaker Intl1 binding site. Mutation of the stronger binding site showed that a single base pair change accounted for the difference in the strength of binding.