Annotation of the honey bee genome has revealed that it encodes 47 cytochrome P450 monooxygenases (P450s), a superfamily of enzymes that metabolize xenobiotics and synthesize and degrade insect hormones. This number is far fewer than that reported for P450s encoded in the Anopheles gambiae (111) or Drosophila melanogaster (89). Honey bees appear to be lacking in representatives of the insect-specific CYP4 family, with only three CYP4 sequences compared with 22 in D. melanogaster and 29 in A. gambiae. The number of P450s related to ecdysteroid metabolism appears to be well conserved. Five honey bee P450s appear to be orthologous to D. melanogaster P450s in active in the process of converting cholesterol to 20-hydroxyecdysone, the active molting hormone. Involvement of these orthologs in ecdysteroid synthesis was tested by comparing mRNA expression in adult queens, which have high titers of ecdysone, and adult workers, which produce no ecdysone. The function of CYP314A1, ortholog to the 20-hydroxylase converting ecdysone to 20-hydroxyecdysone in D. melanogaster, was examined by baculovirus protein expression. Evidence from this study suggests that P450s related to hormone biosynthesis may show a higher degree of conservation in number as well as sequence and function across genomes than those involved in xenobiotic metabolism.