Twelve monoclonal antibodies (MAbs) to rat cytochrome P450s and one MAb to a scup (fish) P450 have been isolated, characterized, and are currently in common use. Expression of cDNAs for different P450s from a vaccinia vector offers a rapid and simple way toward the production of individual P450s. The thirteen MAbs were examined for their cross-reactivity with ten cDNA expressed human, rat, and mouse P450s. Three MAbs to rat 1A1 and fish 1A1 cross-reacted with cDNA expressed mouse 1A1. One of the latter MAbs, 1-7-1 but none of the others cross-reacted with mouse 1A2. Surprisingly, the fish MAb to 1A1 also cross-reacted with human 2E1. Two MAbs to rat 2B1/2B2 cross-reacted with rat 2A1. An MAb to rat 2C11 cross-reacted with human 2C9. Two MAbs to rat 2E1 cross-reacted with human 2E1. Finally, two MAbs to rat 3A1 cross-reacted strongly with human 3A4. These studies open the door to constructing a library of MAbs with defined binding activity to the P450s of human and other species.