The human intestinal di/tripeptide transporter hPepT1 (when expressed heterologously in Xenopus laevis oocytes or HeLa cells) functions as an H⁺-coupled, pH-dependent, Na⁺-independent transporter (Liang et al. 1995; Kennedy et al. 2002). In contrast, optimal dipeptide uptake in intact intestinal epithelia is partially dependent on extracellular Na⁺ as a result of the functional coupling between hPepT1 and the Na⁺/H⁺ exchanger NHE3 (Kennedy et al. 2002). 5-(N-ethyl-N-isopropyl)amiloride (EIPA), which is used commonly to inhibit Na⁺/H⁺ exchange, reduces dipeptide uptake across the apical membrane of Caco-2 cell monolayers even in the absence of NHE3 activity (Na⁺-free conditions) (Kennedy & Thwaites, 2003). At concentrations higher than those required to inhibit Na⁺/H⁺ exchange, amiloride and analogues interact with a number of other transporters (Kleyman & Cragoe 1988). The aim of this study was to identify whether EIPA inhibition of dipeptide uptake is direct on hPepT1 or indirect via a reduction in the transmembrane ionic driving force (the H⁺-electrochemical gradient). X. laevis were killed humanely and oocytes removed. Gly-Sar or proline (both 100μM, 5μCi.ml^-1) uptake (40min, pH_o 6.0, Na⁺-free conditions) was determined in oocytes 3 days after injection with 50ng hPepT1 or rat PAT2 cRNA, respectively. In hPepT1-expressing oocytes, EIPA inhibited Gly-Sar uptake [mean ± SEM (n)] in a concentration-dependent manner and at 500μM EIPA reduced uptake by 68±4 (29) % (p0.05). Under voltage-clamped conditions 500μM EIPA reduced the dipeptide-induced (1mM Gly-Sar) current in hPepT1-injected oocytes. Proline uptake into oocytes expressing the H⁺-coupled amino acid transporter rat PAT2 (Kennedy et al. 2005) was unaffected (p>0.05) in the presence of 500μM EIPA (98±10 (18) % of control, absence of EIPA). In conclusion, high concentrations (500μM) of EIPA inhibit dipeptide uptake via a Na⁺-independent, non-NHE3 mediated pathway. This inhibition is likely through a direct effect on hPepT1 rather than an indirect effect on the H⁺-electrochemical gradient as EIPA is without effect on H⁺-coupled amino acid uptake via rat PAT2.