Previous work on Drosophila santomea suggested that its absence of abdominal pigmentation, compared to the other darkly pigmented species, is based on mutations in the cis-regulatory region of tan, inactivating the expression of that gene in the abdomen of D. santomea males and females. Our discovery that D. santomea males can produce viable hybrids when mated to D. melanogaster females enables us to use the armamentarium of genetic tools in the latter species to study the genetic basis of this interspecific difference in pigmentation. Hybridization tests using D. melanogaster deficiencies that include tan show no evidence that this locus is involved in the lighter pigmentation of D. santomea females; rather, the pigmentation difference appears to involve at least four other loci in the region. Earlier results implicating tan may have been based on a type of transgenic analysis that can give misleading results about the genes involved in an evolutionary change.