The purpose of this study was to investigate labetalol as a potential high permeability reference standard for the application of Biopharmaceutics Classification Systems (BCS). Permeabilities of labetalol and metoprolol were investigated in animal intestinal perfusion models and Caco-2 cell monolayers. After isolating specific intestinal segments, in situ single-pass intestinal perfusions (SPIP) were performed in rats and mice. The effective permeabilities (P-eff) of labetalol and metoprolol, an FDA standard for the low/high P-eff class boundary, were investigated in two different segments of rat intestine (proximal jejunum and distal ileum) and in the proximal jejunum of mouse. No significant difference was found between P-eff of metoprolol and labetalol in the jejunum and ileum of rat (0.33 +/- 0.11 X 10(-4) vs 0.38 +/- 0.06 X 10(-4) and 0.57 +/- 0.17 X 10(-4) vs 0.64 +/- 0.30 X 10(-4) cm/s, respectively) and in the jejunum of mouse (0.55 +/- 0.05 X 10(-4) vs 0.59 +/- 0.13 X 10(-4) cm/s). However, P-eff of metoprolol and labetalol were 1.7 and 1.6 times higher in the jejunum of mouse, compared to the jejunum of rat, respectively. Metoprolol and labetalol showed segmental-dependent permeability through the rat intestine, with increased P-eff in the distal ileum in comparison to the proximal jejunum. Most significantly, P-eff of labetalol was found to be concentration-dependent. Decreasing concentrations of labetalol in the perfusate resulted in decreased P-eff compared to P-eff of metoprolol. The intestinal epithelial permeability of labetalol was lower than that of metoprolol in Caco-2 cells at both apical pH 6.5 and 7.5 (5.96 +/- 1.96 X 10(-6) VS 9.44 +/- 3.44 X 10(-6) and 15.9 +/- 2.2 X 10(-6) vs 23.2 +/- 7.1 X 10(-6) cm/s, respectively). Labetalol exhibited higher permeability in basolateral to apical (BL-AP) compared to AP-BL direction in Caco-2 cells at 0.1 times the highest dose strength (HDS) (46.7 +/- 6.5 X 10(-6) vs 14.2 +/- 1.5 X 10(-6) cm/s). The P-eff inhibitor, verapamil, significantly increased AP-BL and decreased BL-AP direction transport of labetalol. Overall, labetalol showed high P-eff in rat and mouse intestinal perfusion models similar to metoprolol at a concentration based on HDS. However, the concentration-dependent permeability of labetalol in mice due to P-gp and the inhibition study with verapamil in Caco-2 cells indicated that labetalol is not an ideal reference standard for BCS classification.