- Development of folate deficiency (FD) was evaluated in weanling rats fed diets containing mixtures of free amino acids or of vitamin-free casein and gelatin as sources of dietary nitrogen. FD could be produced in 21 d with amino acid diets that promoted maximum growth rate, were completely devoid of folate and contained 1% succinylsulfathiazole. Growth retardation and blood dyscrasia associated with FD could not be demonstrated in rats fed diets containing casein and gelatin as nitrogen sources because the vitamin-free casein contained low but measurable levels of folate. The most effective protocol to produce experimental FD in rats is to feed a folate-free diet that otherwise supports maximum growth in young animals. Additional modifications such as use of methotrexate or amino acid-imbalanced or protein-deficient diets are unnecessary.