Intestinal epithelium is a complex organ that undergoes continuous proliferation. D-type cyclins bind cyclin-dependent kinases (Cdk4 and Cdk6) and are expressed during the transition from G0 into the S phase. Previously, we reported that cyclins D1 and D3 are induced by growth factors in two rat intestinal epithelial cell lines, IEC-6 and RIE-1. However, transforming growth factor beta induces G1 arrest in both intestinal cell lines without inhibiting cyclin D3, suggesting that cyclin D3 may not have essential functions in the gut. In the present study, we determined whether cyclin D3 is required for the transition from G0 into the S phase in intestinal epithelial cells. Microinjection of anti-cyclin D3 antiserum inhibited quiescent IEC-6 and RIE-1 cells from entering the S phase, while cells microinjected with a nonspecific mouse immunoglobin G continued to progress into the S phase. We also examined the expression of cyclin D3 in rat jejunal mucosa after fasting and refeeding. Cyclin D3 levels were not altered by fasting and refeeding; however, Cdk4 expression was suppressed by fasting and returned to control levels after refeeding. Our results suggest that cyclin D3 is essential for intestinal epithelial cell proliferation, although its expression is not regulated by dietary restriction.