The human intestinal proton-coupled oligopeptide transporter hPEPT1 has been implicated in the absorption of pharmacologically active compounds. We have investigated the interactions between a comprehensive selection of drugs, and wild-type and variant hPEPT1s expressed in Xenopus oocytes, using radiotracer uptake and electrophysiological methods. The β-lactam antibiotics ampicillin, amoxicillin, cephalexin and cefadroxil, the antineoplastics δ-aminolevulinic acid (δ-ALA) and bestatin, and the neuropeptide N-acetyl-Asp-Glu (NAAG), were transported, as judged by their ability to evoke inward currents. When the drugs were added in the presence of the typical substrate glycylsarcosine (Gly-Sar), the inward currents were equal or less than that induced by Gly-Sar alone. This suggests that the drugs are transported at a lower turnover rate than Gly-Sar, but may also point towards complex interactions between dipeptides, drugs and the transporter. Gly-Sar and the drugs also modified the kinetics of hPEPT1 presteady-state charge movement, by causing a reduction in maximum charge (Qmax) and a shift of the midpoint voltage (V0.5) to more negative potentials. Our results indicate that the substrate selectivity of hPEPT1 is: Gly-Sar > NAAG, δ-ALA, bestatin > cefadroxil, cephalexin > ampicillin, amoxicillin. Based on steady-state and presteady-state analysis of Gly-Sar and cefadroxil transport, we proposed an extension of the 6-state kinetic model for hPEPT1 function that globally accounts for the observed presteady-state and steady-state kinetics of neutral dipeptide and drug transport. Our model suggests that, under saturating conditions, the rate-limiting step of the hPEPT1 transport cycle is the reorientation of the empty carrier within the membrane. Variations in rates of drug cotransport are predicted to be due to differences in affinity and turnover rate. Oral availability of drugs may be reduced in the presence of physiological concentrations of dietary dipeptides in the gut, suggesting that oral delivery drugs should be taken on an empty stomach. The common hPEPT1 single-nucleotide polymorphisms Ser117Asn and Gly419Ala retained the essential kinetic and drug recognition characteristics of the wild type, suggesting that neither variant is likely to have a major impact on oral absorption of drugs.