1. The aim was to investigate the molecular characterisation and effects of dietary folate supplementation on mRNA concentrations of the proton-coupled folate transporter (PCFT) in chicken. 2. Twenty-four (n = 8/treatment) laying hens were allocated at random to one of three dietary treatments: a basal diet without supplemental folate, or the same diet with either 10·00 mg/kg folic acid or 11·30 (equimolar) mg/kg 5-methyltetrahydrofolate (5-MTHF) for 21 d. The PCFT cDNA was cloned using the TA cloning system, and mRNA concentrations were determined by real-time polymerase chain reaction (PCR). 3. The PCFT cDNA (1643 bp) was cloned from the duodenum and its transcripts were widely distributed in chicken tissues. Duodenal and caecal PCFT mRNA was not affected by dietary treatments; however, jejunal PCFT mRNA was significantly reduced in hens fed the 5-MTHF diet versus the basal diet. 4. A broad tissue distribution of PCFT transcripts is indicative of a vital role of PCFT in the folate transport in chicken. The expression of jejunal PCFT mRNA was down-regulated by dietary 5-MTHF supplementation. These findings contribute to an understanding of the development of folate transport and the PCFT system in avian systems involving laying hens. © 2010 British Poultry Science Ltd.