The NADPH oxidases are a group of plasma membrane-associated enzymes found in a variety of cells. They catalyze the production of superoxide (O(-)(2)) by a one-electron reduction of oxygen, using NADPH as the electron donor. To characterize the expression of this enzyme, two homologues of the NADPH oxidase catalytic subunit, gp91(phox), were cloned from the cDNAs of a human colon cancer cell line, Caco2, and human fetal kidney, using information relating to an expressed sequence tag (EST) from a DNA database. Amino acid identity was 58% (gp91-2) and 56% (gp91-3), respectively, against the catalytic subunit (gp91-1/gp91(phox)) of the NADPH oxidase found in peripheral blood leukocytes. Using the reverse transcription-polymerase chain reaction (RT-PCR) method, the messenger RNA of gp91-2 was detected mainly in the colon (and also in kidney and prostate) among human adult tissues, in the thymus among human fetal tissues, and in the cancer cell lines (HepG2 and Caco2). An expression of gp91-3 was detected in the fetal kidney, and in the cancer cell line (HepG2), but not at all in adult tissues (by the RT-PCR method). In situ hybridization revealed that gp91-2 is located in the absorptive epithelial cells of the adult colon. Neither gp91-2 nor gp91-3 was expressed in peripheral blood leukocytes.