Peptide transporter 1, PEPT1, of the mammalian enterocyte is presently under intense investigation in many laboratories because of its nutritional importance in the absorption of protein hydrolysis products and because more recent studies have shown that many drugs and prodrugs gain entry into the systemic circulation via PEPT1. Until the exact structural features of the substrate binding site of PEPT1 become available, for example by X-ray crystallography, determination of affinities followed by proof of actual membrane translocation will have to suffice when testing for possible new substrates for PEPT1. Affinity constants reflect the strength of their interaction with the binding site of the transporter. A review of the literature shows a wide range of affinity constants between 2 microM and 30 mM. We consider affinity constants for substrates or inhibitors of PEPT1 lower than 0.5 mM as high affinity, between 0.5 and 5.0 mM as medium affinity and above 5 mM as low affinity. Values above 15 mM we consider with great caution. In this mini-review we discuss affinities and structural determinants which affect affinities of a variety of substrates for PEPT1.