We have recovered two Hessian fly cytochrome P450 cDNAs from an ongoing midgut EST project. CYP6AZ1 and CYP6BA1 represent two new subfamilies within the CYP6 family. The deduced amino acid sequences for CYP6AZ1 and CYP6BA1 show conserved structural and functional domains of insect P450s. Expression analysis with reverse transcription-polymerase chain reaction (RT-PCR) indicated that CYP6AZ1 is midgut specific and induced during active larval feeding, whereas CYP6BA1 was expressed in all tissues and developmental stages examined. Further expression analysis of CYP6AZ1 with RT-PCR in compatible and incompatible Hessian fly/wheat interactions suggested that CYP6AZ1 may be required for larval feeding in compatible interactions. These results should lead to a better understanding of the Hessian fly/wheat interaction with emphasis on the larval midgut as a critical interface with its host plant.