We cloned two novel cytochrome P450 cDNAs (CYP2D23 and CYP2D24) from a rabbit liver cDNA library. The open-reading frames of these cDNAs encode proteins that are each composed of 500 amino acids. The amino acid sequence identity of CYP2D23 with CYP2D24 is 91.6%, and the homology of these two isozymes with other known mammalian CYPs in the CYP2D subfamily range from 64.9 to 79.8%. Using RT-PCR, we determined the distribution of these two isozymes in 9 major organs, including brain tissue sections. CYP2D23 mRNA was abundantly expressed in the liver and small intestine, but only slightly in the brain sections, whereas CYP2D24 mRNA was expressed in the liver, small intestine, and stomach. CYP2D23 and CYP2D24 were heterogeneously expressed in 293T cells. CYP2D24 effectively catalyzed the oxidation of bufuralol and bunitrolol, the archetypal substrates of the CYP2D subfamily, while CYP2D23 exhibited catalytic activity only toward bufuralol. The results of this first study on rabbit CYP2D isozymes indicate that CYP2D23 and CYP2D24 are functionally expressed in rabbits, and have different organ distributions and metabolic properties.