The intestinal expression of the CYP3A subfamily was investigated in female rats, and the intestinal metabolism of two CYP3A substrates, testosterone and rifabutin, was examined and compared between males and females. CYP3A1/23 and CYP3A2 intestinal expression was barely detected in male and female rats. Although CYP3A9 was predominantly expressed in the female rat liver, its expression in the intestine was not different between the two sexes. The rate of testosterone 6β-hydroxylation in the female intestine was similar to that for males. Rifabutin was also metabolized at similar rates in both intestines, although the metabolic rate was greater in the female liver. These results indicate that the intestinal drug metabolizing activity of the CYP3A subfamily is similar between males and females, and that CYP3A9 is involved in the intestinal metabolism of CYP3A substrates in both sexes.