The insect molting hormone, 20-hydroxyecdysone (20E), is a major modulator of the developmental processes resulting in molting and metamorphosis. During evolution selective forces have preserved the Halloween genes encoding cytochrome P450 (P450) enzymes that mediate the biosynthesis of 20E. In the present study, we examine the phylogenetic relationships of these P450 genes in holometabolous insects belonging to the orders Hymenoptera, Coleoptera, Lepidoptera and Diptera. The analyzed insect genomes each contains single orthologs of Phantom (CYP306A1), Disembodied (CYP302A1), Shadow (CYP315A1) and Shade (CYP314A1), the terminal hydroxylases. In Drosophila melanogaster, the Halloween gene spook (Cyp307a1) is required for the biosynthesis of 20E, although a function has not yet been identified. Unlike the other Halloween genes, the ancestor of this gene evolved into three paralogs, all in the CYP307 family, through gene duplication. The genomic stability of these paralogs varies among species. Intron-exon structures indicate that D. melanogaster Cyp307a1 is a mRNA-derived paralog of spookier (Cyp307a2), which is the ancestral gene and the closest ortholog of the coleopteran, lepidopteran and mosquito CYP307A subfamily genes. Evolutionary links between the insect Halloween genes and vertebrate steroidogenic P450s suggest that they originated from common ancestors, perhaps destined for steroidogenesis, before the deuterostome-arthropod split. Conservation of putative substrate recognition sites of orthologous Halloween genes indicates selective constraint on these residues to prevent functional divergence. The results suggest that duplications of ancestral P450 genes that acquired novel functions may have been an important mechanism for evolving the ecdysteroidogenic pathway. © 2007 Elsevier B.V. All rights reserved