The intestinal proton-coupled peptide transporter PepT1 mediates the uptake of di- and tri-peptides and peptidomimetic drugs in the intestine and kidney (reviewed by Meredith & Boyd, 2000). PepT1 has 12 transmembrane spanning regions, and it has previously been proposed that certain transmembrane(TM) tyrosines contribute to the transport function of human PepT1 (hPepT1: Y12 (TM1) and Y167 (TM5), Bolger et al. 1998; Y167, Yeung et al. 1998; Y56 and Y64 (both TM2), Chen et al. 2000). There are a further five TM tyrosines conserved between the mammalian PepT1 sequences: Y91 (TM3), Y287 (TM7), Y345 (TM8), Y587 (TM10) and Y648 (TM12). Here we report the results from a systematic investigation of their functional role in rabbit PepT1. Individual tyrosine residues were site-directed mutagenised to phenylalanine in a rabbit FLAG epitope-tagged PepT1, the mutant PepT1 constructs expressed in Xenopus laevis oocytes, and the uptake of the neutral dipeptide [³H]-D-Phe-L-Gln (0.4μM) measured (Panitsas et al, 2004). Data are mean ± SEM of ‘n’ oocyte preparations (5 oocytes per data point in each). The Y91F mutant showed a significant decrease in [³H]-D-Phe-L-Gln uptake (31.6 ± 5.7% of the wild-type, p<0.05, student's t-test) compared to the wild-type at pHout 5.5 (Figure 1A). In contrast, the Y287F, Y345F, Y587F and Y648F PepT1 mutants showed no apparent variation in uptake compared to the wild-type transporter. The Y12F and Y167F mutants gave similar results to those in the literature, with the exception of a measurable activity of Y167F (6.8 ± 0.9% of wild-type, compared to an abolished activity in HEK 293 cells, Yeung et al. 1998). Surface expression quantification by luminometry for Y12F, Y91F and Y167F suggested that the diminished uptake of D-Phe-L-Gln was not due to an effect on protein synthesis or trafficking (data not shown). Computer modeling studies have predicted that Y91 had a role in proton interaction with hPepT1 (Bolger et al. 2000). To assess whether the tyrosine was important in the proton coupling of peptide transport, uptakes were performed at pHout 5.5, 7.4 and 8.4 (Figure 1B). For both Y91F and Y167F the pH dependence of uptake was similar to that of the wild-type PepT1. In summary, we conclude that Y12, Y91 and Y167 play an important functional role in peptide transport by rabbit PepT1, as the mutant proteins showed reduced uptake rates. The exact mechanism by which this reduction in transport occurs still remains to be elucidated.