Aims: The fluorophore-conjugated dipeptide derivative D-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (AMCA) has been used to visualize dipeptide transport in renal-derived LLC-PK1 cells, rat kidney tubules, mammalian enteric nervous system, all expressing the high-affinity renal type peptide transporter PEPT2, as well as in C. elegans intestinal cells expressing PEPT1 and E. coli expressing YdgR, the worm and prokaryotic homologues of the low-affinity mammalian intestinal transporter PEPT1. However, AMCA uptake by PEPT1 into vertebrate intestinal cells is still a question. Methods: This prompted us to investigate the transport of AMCA in Xenopus oocytes expressing PEPT1- and PEPT2-type transporters using the two-electrode voltage clamp system. In addition, fluorescence of oocytes incubated in AMCA-containing solutions was measured in the absence and presence of the dipeptide substrate glycyl-glutamine (GQ). Results: High GQ-inhibitable fluorescence was observed in oocytes expressing worm (ce) PEPT1 (907±174 arbitrary units), rabbit (r) PEPT2 (1260±53), zebrafish (zf) PEPT2 (1270±255) and, to a somewhat lesser degree, in oocytes expressing the chimeric transporter rPEPT2/1 from rabbit PEPT2 (N-terminal part) and PEPT1 (C-terminal part) (514±51). No AMCA transport activity was observed in cePEPT2 (the high-affinity transporter), rPEPT1, zfPEPT1 and rPEPT1/2 expressing oocytes. AMCA substrate affinity (mM) and transport rate (percent of GQ transport) were 2.2±0.2 and 9±1% (cePEPT1), 0.29±0.03 and 35±2% (rPEPT2) and 0.30±0.03 and 52±3% (zfPEPT2). Conclusion: These results show that the heavy fluorophore attached to the side chain of lysine hinders its translocation in mammalian PEPT1-type, but not in PEPT2-type transporters. This limitation is probably raised by the N-terminal part of the transporter. In an invertebrate AMCA transport was observed in a PEPT1-homologue transporter suggesting distinct features in the substrate binding domains.