Microsomal fractions from mouse lymphoma L5178Y and from rat thymocytes were used to follow incorporation of radiolabel from cytidine diphosphate-[methyl-14C]choline into microsomal lipids. Dexamethasone, at concentrations ranging from 2.8 X 10(-6)M to 2.8 X 10(-5)M, partially inhibited this transfer reaction. Microsomes prepared from freshly isolated thymocytes were more sensitive to the effects of dexamethasone showing inhibition at concentrations of steroid as low as 2.8 X 10(-8)M. The inhibitory effect did not depend on the amount of the available endogenous diglycerides and was not related to a possible stimulation of cytidine diphosphate choline transferase back reaction by the steroid. The survey of a broad selection of different steroids revealed a lack of correlation between the known lymphocytolytic properties of steroids and their effects on cytidine diphosphate choline transferase. Dexamethasone was the only steroid of the glucocorticoid group that inhibited this reaction in microsomal fractions of L5178Y lymphoma. The structural requirement for the inhibitory effect was related to the absence of oxygen functions in positions 11 and 17 of the steroid and, possibly, to the presence of both C-20 and C-21 on the side chain.