Cynomolgus macaques are frequently used in drug metabolism studies due to their evolutionary closeness to humans. Despite their importance, genes encoding drug-metabolizing enzymes have not been fully identified in this species. In this study, the cDNA orthologous to human cytochrome P450 3A43 (CYP3A43) was isolated. Deduced amino acids of this cDNA had a high sequence identity ( approximately 95%) to human CYP3A43 cDNA and contained characteristic motifs for CYP3A proteins, heme-binding region and substrate recognition sites. Among 10 tissues analyzed, cynomolgus CYP3A43 was expressed in liver, adrenal gland, and lung, with the highest expression seen in liver. Cynomolgus CYP3A43 protein heterologously expressed in Escherichia coli exhibited metabolic activity toward midazolam 1'-hydroxylation. These results indicated that cynomolgus CYP3A43 was expressed in liver and encoded a functional drug-metabolizing enzyme, and could contribute to overall drug metabolism in cynomolgus macaque liver if expressed as a protein.