House fly cytochrome P450 CYP6D1 carries out the metabolism of xenobiotics. Expression of house fly CYP6D1 is induced in response to the prototypical P450 inducer, phenobarbital (PB), in the insecticide susceptible strains, CS and aabys. In the permethrin resistant LPR strain, increased transcription of CYP6D1 confers the metabolism-mediated resistance. CYP6D1 is constitutively overexpressed without significant PB induction in LPR. A series of experiments were conducted to understand the transcriptional regulation of CYP6D1. The core promoter of CYP6D1 is a dispersed type, as two transcription start sites were identified. Assays of the CYP6D1v2 promoter from the CS strain in Drosophila S2 cells identified promoter regions critical for basal transcription and for PB induction. Using RNAi treatment of Drosophila S2 cells, HR96 (hormone receptor-like in 96) and BR-C (broad-complex) were identified to be transcription factors critical for PB induction of CYP6D1v2. HR96 and BR-C were an activator and repressor, respectively, of PB induction of CYP6D1v2. The same promoter region of CYP6D1v1 from LPR was examined and shown to mediate PB induction to similar levels as CYP6D1v2 from CS. This indicates variations in promoter sequences are not responsible for the lack of PB induction of CYP6D1v1. Therefore, constitutive overexpression without PB induction of CYP6D1 in LPR is due to an unidentified trans acting factor. HR96 was cloned and sequenced to examine if it is this trans acting factor. Multiple HR96 alleles were identified and alleles v8-v10 were found to encode E28V and G110D amino acid substitutions in LPR. Permethrin selection of LPR showed HR96 alleles v8-v10 were not associated with permethrin resistance. Quantitative real-time RT-PCR showed no difference of HR96 expression levels between LPR and CS. Thus HR96 is not the trans acting factor responsible for constitutive overexpression of CYP6D1 in LPR. The molecular basis of constitutive overexpression of CYP6D1 in LPR is attributed to a trans acting factor responsible for PB induction in susceptible strains, but this trans acting factor remains unidentified.