(R)- and (S)-dichlorprop/alpha-ketoglutarate dioxygenases (RdpA and SdpA) catalyze the oxidative cleavage of 2-(2,4-dichlorophenoxy)propanoic acid (dichlorprop) and 2-(4-chloro-2-methyl-phenoxy)propanoic acid (mecoprop) to form pyruvate plus the corresponding phenol concurrent with the conversion of alpha-ketoglutarate (alphaKG) to succinate plus CO2. RdpA and SdpA are strictly enantiospecific, converting only the (R) or the (S) enantiomer, respectively. Homology models were generated for both enzymes on the basis of the structure of the related enzyme TauD (PDB code 1OS7). Docking was used to predict the orientation of the appropriate mecoprop enantiomer in each protein, and the predictions were tested by characterizing the activities of site-directed variants of the enzymes. Mutant proteins that changed at residues predicted to interact with (R)- or (S)-mecoprop exhibited significantly reduced activity, often accompanied by increased Km values, consistent with roles for these residues in substrate binding. Four of the designed SdpA variants were (slightly) active with (R)-mecoprop. The results of the kinetic investigations are consistent with the identification of key interactions in the structural models and demonstrate that enantiospecificity is coordinated by the interactions of a number of residues in RdpA and SdpA. Most significantly, residues Phe171 in RdpA and Glu69 in SdpA apparently act by hindering the binding of the wrong enantiomer more than the correct one, as judged by the observed decreases in Km when these side chains are replaced by Ala.