Maintenance of epithelial differentiation is one of the biological functions of vitamin A and explains current interest in this nutrient as a chemopreventative agent of epithelial cancer. Therefore, current research efforts center on the elucidation of possible sites and mechanisms of action of the vitamin. These were pursued at the following levels: 1) Protein glycosylation: under conditions of vitamin A deficiency, the in vivo incorporation of mannose into guanosine diphosphomannose, dolichylphosphatemannose and lipid-linked oligosaccharides was markedly decreased and resulted in the accumulation of shorter oligosaccharides on lipid-linked intermediates involved in protein glycosylation. At the same time an accumulation of mannose and dolichylphosphate was also observed, suggesting that vitamin A regulates protein glycosylation by controlling the level of GDP-mannose and dolichylphosphate mannose in the membrane. During this work a new mannolipid, phosphatidyl-mannose, was found to be synthesized in vitro by liver microsomal membranes. 2) Keratin gene expression: we have employed a tracheal organ culture system and have reproduced the in vivo phenomenon of squamous metaplasia during culturing under vitamin A free conditions as well as after carcinogen treatment. Vitamin A deficient tracheas were shown by immunoblot analysis to contain keratins of 50, 48, 46.5 Kd detected with the antibody AE1, and 58, 56, and 52 Kd detected with AE3. These proteins were either absent or present in much less quantity in control tracheas. In deficient tracheas 60 Kd keratin was found to be located specifically in squamous suprabasal cells, and 55 and 50 Kd keratin proteins were found in a basal cell compartment. Following carcinogen exposure, the appearance of 60 Kd keratin and the enhanced expression of 50 and 55 Kd keratins preceded the squamoid metaplastic response as detected morphologically. Both the keratin changes and the morphological changes were prevented by retinoid treatment. 3) Retinoid transport: a new assay to separate the cellular retinol binding protein from the cellular retinoic acid binding protein by HPLC on Mono Q columns was developed.