An earlier study of cholesterol metabolism in HeLa cells has provided evidence for the inhibitory effect of glucocorticoids on de novo cholesterol synthesis. To extend this observation to other regulatory pathways involving cholesterol, we have now determined the effects of dexamethasone on the cell-associated low density lipoprotein (LDL) assumed to represent a normal source of exogenous cholesterol. It was found that HeLa cells internalized LDL by low affinity as well as high affinity saturable processes. Since cell-associated LDL was reduced by dexamethasone to 20-60% of the control value, we have made further attempts to examine the nature of this observation. The kinetics of LDL-cell association and the binding studies performed at 4 C indicated that dexamethasone did not affect the immediate binding of LDL to HeLa cells, whereas 25-hydroxycholesterol, which is known in other cells to inhibit LDL binding by reducing the number of LDL receptors, had the same effect in HeLa cells. The nature of the dexamethasone effect appeared to be related to interference with processing of bound LDL after its internalization. Further evidence that dexamethasone did not affect the initial binding of LDL was provided by the experiments in which chloroquine completely abolished the inhibitory action of dexamethasone.