Toluate dioxygenase of Pseudomonas putida mt-2 (TADO(mt2)) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADO(ADP1)) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the alpha subunit of one enzyme and the beta subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADO(ADP1) utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADO(mt2) (3-methylbenzoate > benzoate approximately 3-chlorobenzoate > 4-methylbenzoate approximately 4-chlorobenzoate >> 2-methylbenzoate approximately 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the alphaBbetaT hybrid oxygenase for these benzoates corresponded to that of BADO(ADP1), the parent from which the alpha subunit originated. In contrast, the apparent substrate specificity of the alphaTbetaB hybrid oxygenase differed slightly from that of TADO(mt2) (3-chlorobenzoate > 3-methylbenzoate > benzoate approximately 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the alphaTbetaB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADO(mt2) parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O2 utilization in the hybrid than in the parent. Overall, these results support the notion that the alpha subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the beta subunit contributes significantly to the enzyme's function.