In cultured human fibroblasts, each LDL receptor mediates the internalization of approximately 100 particles of LDL every 20 hr. We provide evidence that this reutilization of LDL receptors involves the recycling of receptors into and out of the cell and that the carboxylic ionophore monensin blocks the return of the receptors to the surface. In the presence of monensin and LDL, 75% of the receptors disappeared from the cell surface within 15 min and more than 90% disappeared within 60 min. The receptors that left the surface were trapped intracellularly within perinuclear vacuoles, as visualized by indirect immunofluorescence with the use of LDL, monensin caused about 50% of the receptors to be trapped intracellularly within 15 min. The receptors that remained on the surface after monensin treatment could be trapped within the cell if LDL was added subsequently in the continued presence of monensin. Monensin did not decrease surface LDL receptors in fibroblasts from a patient (J.D.) with the internalization-defective form of familial hypercholesterolemia. In these mutant cells, LDL receptors are not localized to coated pits. The current data are interpreted to indicate that: in normal fibroblasts about 50% of surface LDL receptors absence of LDL; the remaining 50% of surface receptors can be induced to recycle by the presence of LDL; and monensin interrupts this recycling by preventing the receptor from returning to the surface, thereby causing the receptors to accumulate within the cell.