Polychlorinated biphenyls (PCBs) are persistent organic pollutants. Biphenyl 2,3-dioxygenase (BDO) is a key enzyme that determines the range of PCBs oxidized by a bacterial strain. BDO subunit α (BphA1) plays an essential role in substrate recognition and binding. The genes for dioxygenases that hydroxylate aromatic rings were screened and analyzed phylogenetically. Genes found in biphenyl-oxidizing Rhodococcus erythropolis strains G12a, B7b, and B106a proved to be similar to the published nucleotide sequences of the Rhodococcus sp. HA99 and R04 and Novosphingobium aromaticivorans F199 bphA1 genes, which code for the α-subunits that do not belong to the biphenyl/toluene dioxygenase (B/TDO) family. PCB-destructing R. ruber P25 was found to possess a unique bphA1 gene, which clusters together with the phenylpropionate dioxygenase (PPDO) α-subunits of Mycobacterium vanbaalenii PYR-1 and Frankia sp. EuI1c. The deduced amino acid sequences of the genes were analyzed. The amino acids of the BDO active site in R. wratislaviensis P1, P12, P13, and P20 (bphA1 genes of the B/TDO family) were identical to those of the active PCB degrader R. jostii RHA1. The Rhodococcus strains in question were shown to be active toward both orthoand parachlorinated ring of 2,4'-dichlorobiphenyl. The α-subunit amino acids responsible for the substrate specificity of the enzyme in Pseudomonas sp. S9, S13, S210, S211, and S212 (B/TDO family) were the same as in P. pseudoalcaligenes KF707. The Pseudomonas strains were active toward the para-chlorinated ring of 2,4'-dichlorobiphenyl. The results of screening bacterial strains for bphA1 can be used to identify the biotechnologically promising PCB destructors.