We developed an assay for galactosyl transferase (uridine diphosphategalactose:glycoprotein galactosyltransferase, EC 2.4.1.22) in microsomes of rat tracheal epithelium and characterized the properties of this enzyme system. We found that vitamin A deficiency greatly depressed galactosyl transferase activity. Activity could be restored within 48 h by dosing the animal with retinyl acetate. Adding retinol to the microsomes from tracheal epithelium of vitamin A-deficient rats also restored galactosyl transferase activity to some extent. Full restoration was achieved by pre-incubating retinol with the microsomal preparation for 30 min. Optimal concentration of pre-incubated retinol was 10(-8) M. Pre-incubation with retinyl phosphate and retinoic acid stimulated galactosyl transferase activity in microsomes from tracheas of deficient rats; pre-incubation with dolichyl phosphate, anhydroretinol and the dimethylacetylcyclopentenyl analog of retinoic acid did not. We concluded that vitamin A is involved in the galactosyl transferase system of rat tracheal epithelium, possibly by being linked to galactose.