The membrane transport of glucose was studied in bovine adrenal chromaffin cell cultures by following the cell/medium distribution of the nonmetabolizable glucose analog, 3-O-methyl-D-glucose. Uptake of this sugar in day-1 cultures that are undergoing rapid morphological change and differentiation had a Vmax of 138 nmol/(mg protein.min) and Km of 15 mM, and was only slightly increased by 50 mU/mL insulin. In day-5 cultures where morphological changes were essentially completed, Vmax and Km decreased to 51 nmol/(mg protein.min) and 9.5 mM, respectively, and the response to insulin was restored to the level found in freshly isolated cells; this effect was abolished in the nominal absence of Ca2+. Thus, saturation kinetics and insulin and Ca2+ sensitivity of 3-methylglucose uptake observed in freshly isolated cells were maintained in culture. However, the insulin response was almost absent during the initial period of rapid morphological change when sugar transport was strongly stimulated. Culture of chromaffin cells in the presence of dexamethasone did not inhibit the formation of processes, but decreased 3-methylglucose uptake in day-5 cultures by an apparently competitive effect.