Objective: The proton-coupled amino acid transporter 1, PAT1 (SLC36A1), is a nutrient and drug transporter expressed at the luminal surface of both human and rat intestine. The purpose of this study was to investigate the expression of rPat1 mRNA along the length of the rat intestine. Furthermore, the functionality of the transporter i.e. in vitro uptake of a PAT1 substrate was also measured along the intestine. Methods: A two-step RealTime-PCR was used together with the comparative DDC_T method to evaluate the rPat1 mRNA expression. rGapdh was the reference gene. The expression of rPat1 mRNA was measured in segments of rat intestine taken every 5 cm from the duodenum to the colon. Likewise, intestinal samples were taken to investigate the uptake of a PAT1 substrate. The uptake of 0.1-30 mM [³H]-L-proline (1 mCi/ml) was measured for 15-20 min. in HBSS buffers adjusted with HEPES or MES to pH 7.4 or 6.0. The uptake of proline was corrected for extracellular volume and non-specific uptake using [¹⁴C]-mannitol in all experiments. The uptake of 2.5 mM proline was also measured in the presence of 50 mM GABA, gaboxadol, L- valine or 30 mM 5-hydroxy-L-tryptophan. Results: The expression of rPat1 mRNA along the rat intestine showed a bell-shaped distribution. The maximal expression was observed in mid jejenum where the expression was 7 times higher relative to that of the duodenum. The expression level in the colon was only half the level of the duodenum. The uptake of proline along the intestine showed a similar bell-shaped distribution. The proline uptake was: characterized by a K_m of 2.9±0.44 mM, inhibited by GABA, gaboxadol, 5-HTP and valine however it was not dependent on pH of the buffers which might be due to pH gradients within the mucus layer. Conclusion: The rPat1 mRNA is expressed along the rat intestine from duodenum to colon with maximal expression in the mid jejenum. The pattern of rPat1-mediated proline uptake matches the rPat1 mRNA distribution.