1. The distribution of vitamin A was examined in various subcellular fractions of rat liver and bovine retinal pigment epithelium. In rat liver, the major portion of the vitamin is in the cytosol, whereas in pigment epithelium, it is concentrated mainly in the microsomes. The microsomal vitamin A of pigment epithelium is tightly bound to membranes, as shown by the inability to release it except by organic solvent extraction or incubation with Triton X-100. 2. In both tissues, two different forms of cytosol vitamin A could be distinguished by ultracentrifugation. The major portion in liver is in the floating lipid phase and consists mainly of retinyl ester. The remainder (less than 10% of the total) is in the underlying infranatant; about 90% of the vitamin A in this fraction is esterified. By contrast, two-thirds of the vitamin A of pigment epithelial cell cytosol is in the infranatant; it consists of both esterified and unesterified retinol. The floating layer in the pigment epithelial cytosol consists entirely of retinyl ester. 3. These two forms of cytosol vitamin A in the pigment epithelium could also be separated by gel filtration on Sephadex G-100 which yielded two distinct fluorescent peaks. The first, which appeared in the void volume and corresponded in all probability to the floating layer obtained by ultracentrifugation, consisted only of retinyl ester. The second peak, which was eluted in approximately the same position as myoglobin, contained only unesterified retinol. It was abolished completely by preincubation with pronase. These findings support the view that the second peak represents the endogenous retinol-retinol binding protein complex of pigment epithelial cytosol. The fluorescent enhancement of the retinol bound to protein in this peak was about 4--5-fold compared to retinol in organic solvents.