The uptake of [3H]cholesterol from low density lipoprotein (LDL) was studied in LDL receptor-positive and receptor-negative human fibroblasts. In both cell lines the uptake depended upon temperature, time of incubation and the concentration of LDL in the medium. Although the incorporation of 125I-labeled LDL was minimal after 2 h of incubation in the receptor-negative (homozygous familial hypercholesterolemia, FH) cells, the uptake of [3H]cholesterol was only slightly less than that of the receptor-positive (WI-38) cells. With longer periods of incubation, a larger difference in labeled cholesterol incorporation was observed; this appeared to be due to a continued accumulation of the steroid in the WI-38 cells. After 8 and 24 h of incubation, some of the [3H]cholesterol was present as the ester in the WI-38 cells, but not the FH cells. Modified (reduced and methylated) LDL did not enter WI-38 cells by the receptor-mediated pathway during 2 h of incubation, as indicated by 125I uptake. [3H]Cholesterol uptake, however, was not significantly different from modified and unmodified LDL. While experiments indicated that significant amounts of cholesterol moved rapidly from LDL to cultured cells with a dependence on time and LDL concentration, no increase in total cell cholesterol was detected in either cell line. FH cells contained less total cholesterol and had a higher 3H specific activity than the WI-38 cells. These data suggest that there may be important mechanisms in addition to the LDL pathway for the movement of lipids into cells.