The P450IIIA (CYP3A) cytochromes are a major family of enzymes that play an important role in the metabolism of many medications, including cyclosporine A, as well as some dietary xenobiotics, including aflatoxin B1. The purpose of the studies was to detect, localize, and characterize P450IIIA enzymes present throughout the digestive tract. To this end, P450IIIA-specific antibodies were used to examine gut epithelial microsomes and histological tissue sections obtained from the digestive tract of both male and female rats. P450IIIA-related proteins were detected in epithelia throughout the gut; however, the specific proteins expressed appeared to differ among digestive organs and between male and female rats. RNA obtained from the gut epithelia was also analyzed using P450IIIA-specific synthetic oligonucleotides as probes on Northern blots and as primers for the polymerase chain reaction. P450IIIA1, which is a dexamethasone inducible enzyme in liver, was also found to be induced by dexamethasone treatment in epithelia from stomach and jejunum, but not from colon or esophagus. It was concluded that P450IIIA enzymes are present in mature epithelia throughout the gastrointestinal tract. However, expression of the P450IIIA enzymes is influenced by anatomic location and gender.