Enzymes of the cytochrome P450 3A (CYP3A) sub-family are abundant in adult liver and gut and contribute significantly to the first-pass metabolism of many orally administered drugs. The development of CYP3A enzymes with regard to their expression and activity in enterocytic and hepatic microsomes from 1-day-old through to adult male and female rats has been studied. Microsomes were prepared by calcium precipitation. Enzyme expression was assessed semi-quantitatively by Western blotting using rat polyclonal CYP3A2 and 2C11 antibodies and peptide antibodies specific to rat CYPs 3A1, 3A2, 2C12, and 2C13. The formation of 6beta-hydroxytestosterone (6OHT), determined by HPLC, was used as a measure of enzyme activity. Formation of 6OHT by enterocytic microsomes was similar for males and females and showed a sharp increase at weaning. This pattern was mirrored by levels of immunoquantifiable CYP3A2 (CYP3A9), but CYP3A1 followed a more gradual development. CYPs 2C11, 2C12, or 2C13 were not detected in gut microsomes. In contrast, CYPs 3A1, 3A2, 2C11, 2C12, and 2C13 were all expressed in hepatic microsomes. There was no surge in hepatic enzyme expression or hepatic 6OHT formation at weaning, and a marked sex difference in 6OHT formation was apparent from day 25. The surge in gut activity at weaning may be a protective mechanism against ingested toxins.