In Drosophila, the insecticide resistant 91-R strain is an overproducer and susceptible 91-C and ry(506) strains are the underproducers of CYP6A8 mRNA encoded by a cytochrome P450 gene, Cyp6a8. Low expression of Cyp6a8 in the underproducer strains is due to a downregulatory effect of a putative repressor locus, which is thought to be mutant in the overproducer strain. In the present investigation, organization of Cyp6a8 and promoter activity of its upstream DNA were analyzed. Cyp6a8 has two introns of which intron II is similar to the introns of other insect CYP genes with respect to its length and position. Intron I is only 36 bp long and lacks consensus splice sites. It is also in-frame with the CYP6A8 open reading frame. Therefore, inefficient splicing of intron I may produce two isoforms of CYP6A8. Analysis of Cyp6a8 upstream DNA of the overproducer 91-R strain showed that DNA sequences between -199 and -761 bp are required for the highest constitutive and barbital-induced expression of Cyp6a8. This region has six barbie boxes and binding sites for various transcription factors. Promoter activity of the -11/-761 DNA of the overproducer 91-R strain was found to be 4-fold lower in the genome of underproducer ry(506) strain, which is wild type for the putative repressor gene, than in the genome of F1 hybrids of 91-R and ry(506) strains. These results suggest that -11/-761 Cyp6a8 DNA of the 91-R strain can respond to the active repressor present in the hybrid genome and further support our previous findings that overexpression of Cyp6a8 is a result of mutation of a repressor gene rather than mutation of the cis-regulatory sequences.