1. The aim of this study was to investigate the localisation and expression of the epithelial Ca2+ channel TRPV6 (transient receptor potential vanilloid channel type 6) in different intestinal segments and kidney of laying hens during peak lay. 2. Immunohistochemical analysis of the intestine indicated that TRPV6 was localised to the brush-border membranes of the duodenum, jejunum, ileum, caecum, and rectum. Expression was weaker in the rectum, and little or no expression was found in crypt and goblet cells. In addition, TRPV6 mRNA was quantified amongst different intestinal segments, and expression was highest in the duodenum and jejunum. Furthermore, Western blotting indicated that the duodenum expressed the greatest amount of TRPV6 and the rectum the least with the other segments expressing intermediate levels. 3. In the kidney, distinct immunopositive staining for TRPV6 was detected at the apical domain of the distal convoluted tubules (DCT) and medullary connecting tubules (CNT). Interestingly, distribution of TRPV6 extended to the proximal convoluted tubules (PCT). Furthermore, the kidney expressed lower TRPV6 mRNA and protein levels compared with that in the duodenum. 4. In conclusion, the epithelial Ca2+ channel TRPV6 is strongly expressed in the apical cells of the entire intestine and the renal tubules, suggesting that active Ca2+ transcellular transport plays a crucial role in dietary calcium (re)absorption in laying hens.