The objective of this study was to characterize cytochrome P450 (CYP) activities in both intestinal and hepatic microsomes from Wistar and Sprague-Dawley rats. Specific probes for measuring CYP activities were selected using rat recombinant CYP. The intestinal microsome preparation was optimized getting a more relevant and reproducible abundance of CYPs to measure CYP activities. Testosterone, propranolol, diclofenac, and midazolam were determined as specific substrates of rat CYP2C11, CYP2D2, CYP2C6, and CYP3A, respectively. Ethoxyresorufin and pentoxyresorufin were not specific substrates of CYP1A2 and CYP2B1, respectively. Hepatic and intestinal microsomes expressed active CYP1A1, CYP1A2, CYP2B1, and CYP3A2. Only liver expressed active CYP2C6, CYP2C11, and CYP2D2. Wistar liver expressed more active CYP1A and CYP3A2, but less active CYP2B1 than Wistar intestine. Sprague-Dawley liver expressed more active CYP2B1 and CYP3A2, but less active CYP1A than Sprague-Dawley intestine. In conclusion, CYP activities were qualitatively equivalent but not quantitatively in both strains.