Directed mutagenesis has been applied to the cloned genes of barnase and barstar, the extracellular ribonuclease of Bacillus amyloliquefaciens and its intracellular inhibitor, to locate residues involved in the mutual recognition of these two proteins. Arg59 and His102 of barnase and Asp35 and Asp39 of barstar have been so identified. With both Cys40 and Cys82 mutated to alanines, barstar is still produced in high yield and is functional both in vitro and in vivo. Methods devised for determining relative and absolute dissociation coefficients for various combinations of mutant and wild-type proteins have allowed us to determine a dissociation coefficient for the complex of wild-type barnase and barstar of about 10(-13) M, with off and on rate constants of 10(-5) s-1 and 10(8) M-1 s-1, respectively.