1. An experimental model for folate deficiency was established in the rat, which resulted in a decrease of folate in liver, red cells, serum and intestinal mucosa. Development of folate deficiency was associated with diarrhoea, morphological changes in the intestine, and a reduction in total mucosal DNA, RNA, and protein. 2. A technique was established to isolate epithelial cells from all levels of the mucosal villus. The cells from both control and folate deficient animals were viable for a minimum of 40 minutes, as assessed by oxygen consumption, carbon dioxide production, intracellular K+/Na+ ratios, and dye exclusion. 3. Radioautography showed a delay in cell migration time in folate deficiency and RNA/DNA and protein/DNA ratios were greater, especially at the villus-tips. In villus-tip fractions, sucrase activity per cell was sufficiently increased that total gut sucrase was not decreased despite mucosal atrophy. However (Na+-K+)-ATPase activity per villus-tip cell was not increased and total mucosal (Na+ -K)-ATPase was lower. The relationship of these findings to absorptive function are discussed. 4. A new in vitro model was designed to measure the rates of DNA synthesis and relative contributions of de novo and salvage pathways to synthesis in crypt cells. In normal mucosa the de novo pathway was the most important for thymidylate synthesis; its contribution was reduced to a third in folate deficiency. The lowered rate of DNA synthesis in folate deficiency was increased by the provision of thymidine, suggesting that nutrient provision for the salvage pathway can facilitate replication of epithelial cells in folate deficiency. 5. The role of folate in determining the composition and replication of intestinal epithelium is discussed.