The intestinal di/tripeptide transporter hPepT1 plays an important role in the absorption of dietary protein and peptidomimetic drugs (Rubio-Aliaga & Daniel, 2002). For optimal absorptive transport via hPepT1 an active Na⁺/H⁺ exchanger (NHE3) is required at the brush border membrane (Thwaites et al. 2002). NHE3 is activated by the decrease in pH_i associated with hPepT1-mediated H⁺/dipeptide symport. NHE3 extrudes protons and thus maintains the driving force (the transmembrane H⁺ electrochemical gradient) required for further H⁺/dipeptide transport. NHE3 inhibition either by selective pharmacological inhibitors (e.g. S1611) or through activation of the cAMP/protein kinase A pathway (e.g. by vasoactive intestinal peptide) reduces hPepT1 activity indirectly via a reduction in the driving force (Anderson et al. 2003). The aim of this study was to investigate the effects of various phosphodiesterase inhibitors (which raise intracellular cAMP and cGMP levels by inhibiting their breakdown) on hPepT1 function. [¹⁴C]Gly-Sar uptake (0.5μCi.ml^-1, 100μM) across the apical membrane of human intestinal Caco-2 cell monolayers (passage 100-116, 14-19 days post-seeding) was measured over 15min (apical pH 6.5, basolateral pH 7.4) in the presence and absence of various phosphodiesterase inhibitors. Caffeine and theophylline reduced Gly-Sar uptake in a concentration-dependent manner. 5mM caffeine reduced Gly-Sar uptake [mean ± SEM (n)] from 684±19 to 351±11 (17-18) pmol.cm^-2.[15min]^-1 (p<0.001, ANOVA, Bonferroni post test). In contrast, caffeine and theophylline (5mM) did not inhibit Gly-Sar uptake under conditions where NHE3 would be inactive e.g. in the absence of extracellular Na⁺, no difference (p>0.05) in uptake was observed (244±9 and 242±10 (17-18) pmol.cm^-2.[15min]^-1) in the absence and presence of caffeine (5mM), respectively. Pentoxifylline is a phosphodiesterase inhibitor given orally to treat intermittent claudication (Jocoby & Mohler, 2004). Pentoxifylline (5mM) inhibited Gly-Sar uptake by reducing V_max [from 8246±913 to 5369±1167 (6) pmol.cm^-2.[15min]^-1 (p<0.05, Student's t-test)] without effect on affinity [K_m 1.0±0.2 and 1.6±0.6 (6) mM, (p>0.05, Student’s t-test) in the presence and absence of pentoxifylline, respectively]. These observations suggest that hPepT1-mediated absorption of any drug may be reduced (following NHE3 inhibition) by phosphodiesterase inhibitors from diet or those co-administered as therapeutic agents.