- The FDA classifies a drug substance as high-permeability when the fraction of dose absorbed (Fabs) in humans is 90% or higher. This direct correlation between human permeability and Fabs has been recently controversial, since the β-blocker sotalol showed high Fabs (90%) and low Caco-2 permeability. The purpose of this study was to investigate the scientific basis for this disparity between permeability and Fabs. The effective permeabilities (Peff) of sotalol and metoprolol, a FDA standard for the low/high Peff class boundary, were investigated in the rat perfusion model, in three different intestinal segments with pHs corresponding to the physiological pH in each region: (1) proximal jejunum, pH 6.5; (2) mid small intestine, pH 7.0; and (3) distal ileum, pH 7.5. Both metoprolol and sotalol showed pH-dependent permeability, with higher Peff at higher pH. At any given pH, sotalol showed lower permeability than metoprolol; however, the permeability of sotalol determined at pH 7.5 exceeded/matched metoprolol’s at pH 6.5 and 7.0, respectively. Physicochemical analysis based on ionization, pKa and partitioning of these drugs predicted the same trend and clarified the mechanism behind these observed results. Experimental octanol−buffer partitioning experiments confirmed the theoretical curves. An oral dose of metoprolol has been reported to be completely absorbed in the upper small intestine; it follows, hence, that metoprolol’s Peff value at pH 7.5 is not likely physiologically relevant for an immediate release dosage form, and the permeability at pH 6.5 represents the actual relevant value for the low/high permeability class boundary. Although sotalol’s permeability is low at pH 6.5 and 7.0, at pH 7.5 it exceeds/matches the threshold of metoprolol at pH 6.5 and 7.0, most likely responsible for its high Fabs. In conclusion, we have shown that, in fact, there is no discrepancy between Peff and Fabs in sotalol’s absorption; the data emphasize that, if a compound has high fraction of dose absorbed, it will have high-permeability, not necessarily in the jejunum, but at some point along the relevant intestinal regions.