Background: Protein is absorbed primarily as di/tripeptides, which are transported into the enterocyte exclusively by H+/peptide cotransporter 1 (PEPT1). Diurnal changes in expression and function of several other mucosal transporters occur in rat. Diurnal variations in mRNA, protein, and transport function of PEPT1 occur in rat duodenum and jejunum, but not in ileum. Methods: Mucosal levels of mRNA and protein were determined at 9 AM, 3 PM, 9 PM, and 3 AM (n =6 each) by real time RT-PCR and Western blotting, respectively, in rats maintained in a 12-h light/dark room [light 6 AM to 6 PM]; transporter-mediated uptake of dipeptide (Gly-Sar) was also measured by everted sleeve technique. Results: mRNA transcripts of PEPT1 and Gly-Sar uptake varied diurnally in duodenum and jejunum (peak at 3 PM, P <0.05), but not in ileum; maximal uptake was in jejunum. Vmax (nmol/cm/min) was greater at 3 PM and 9 PM compared with 9 AM (3 PM versus 9 AM: 104 versus 62 in duodenum, and 185 versus 101 in jejunum; P <0.03); Km was unchanged across time points or locations. Protein levels varied minimally in jejunum and ileum with peaks at 9 PM and 3 AM. Conclusion: Gene expression and transport function of PEPT1 vary diurnally in duodenum and jejunum in temporal association with nocturnal feeding of rats. [Copyright &y& Elsevier]