Retinal dehydrogenase type 1 (RALDH1) catalyzes the oxidation of all-trans and 9-cis retinal to the respective retinoic acids (RAs), whereas another member of the aldehyde dehydrogenase (ALDH) family, the phenobarbital-induced aldehyde dehydrogenase (PB-ALDH), is very poorly active. We have previously generated chimeras between these 2 enzymes that displayed selectivity for retinal isomers in crude bacterial extracts. Here we have characterized the kinetic properties of the corresponding purified recombinant proteins. The all-trans selective chimera RALDH-131 converted all-trans retinal to all-trans RA with 2.9-fold lower efficiency than the wild-type RALDH1 and had only residual activity with 9-cis retinal. The converse chimera PB-131 was specific for 9-cis retinal, with no residual activity for all-trans retinal. MgCl2 inhibited the activities of RALDH1 and PB-131, but not of RALDH-131, suggesting that amino acids 132-510 in RALDH are necessary for inhibition by MgCl2. These data demonstrate that the chimeric enzymes act as retinal isomer-selective ALDHs, and suggest that these enzymes may be useful to study the roles of cis RA isomers in embryogenesis and differentiation in vivo.