The interaction of the antibacterial phosphonodipeptide alafosfalinwithmammalianH + /peptide cotransporters was studied in Caco-2 cells, expressing the low-affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high-affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [ 14 C]glycylsarcosine withKi values of ± mMand ± mMfor PEPT1 and PEPT2, respectively.