The relationship between vitamin A deficiency and the formation of the sulfatides in brain was investigated in vivo in rats of various ages. By varying litter size and by pair feeding, the control animals had body weights similar to those of the deficient rats. No significant differences in the concentrations or in the total amounts of sulfatides were detected in the brains of vitamin A deficient or control rats, weanling or adult, or in most of the preweanling rats studied at the period of rapid myelination. Except for relatively small, malnourished, vitamin A deficient preweanling rats, variation in body weight within animals of the same age group, did not by itself influence the content of brain sulfatides. Vitamin A deficient animals also showed equal or greater incorporation of (35)S into sulfatides compared to controls. Deficient rabbits were able to incorporate (35)S into the sulfatides of brain and ocular tissues to the same or greater extent as did control animals. Thus, vitamin A deficiency does not interfere in the sulfation of glycolipids or perhaps generally in the processes of biological sulfation as has been proposed previously.