Macrophage cholesterol efflux is important in maintaining cellular lipid homeostasis and preventing the formation of lipid‑laden foam cells. Although radioactive [3H]‑cholesterol is widely used as a tracer in cholesterol efflux assays, the lengthy and labor‑intensive assay procedure, and the radioactivity disposal procedure limit the use of this assay for high‑throughput screening. In the present study, a novel procedure using fluorescent N‑(7‑nitrobenz‑2‑oxa‑1,3‑diazol‑4‑yl)amino)‑23,24‑bisnor‑5‑xholen‑3β‑ol (NBD)‑cholesterol was developed as a substitute for [3H]‑cholesterol for the measurement of cholesterol efflux in THP‑1‑derived macrophages. NBD‑cholesterol uptake and metabolism in the THP‑1 cells were characterized using fluorescent microscopy and spectrophotometry. NBD‑cholesterol distributed rapidly into the cell organelles, with the exception of the nucleus. The uptake of NBD‑cholesterol in the THP‑1 macrophages was concentration‑ and time‑dependent, and reached a plateau following 4 h incubation. The present study subsequently investigated whether NBD‑cholesterol efflux was correlated with [3H]‑cholesterol efflux in THP‑1 derived macrophages and in human peripheral blood mononuclear cells (PBMCs). The results demonstrated that the percentage of efflux of NBD‑cholesterol in the THP‑1 cells was significantly correlated with that of [3H]‑cholesterol, at various concentrations of HDL or apoA‑1 as lipid acceptors (R2=0.876 for HDL; R2=0.837 for apoA‑1; P<0.001). In the PBMCs, NBD‑cholesterol efflux also correlated significantly with [3H]‑cholesterol efflux (R2=0.887 for HDL; R2=0.872 for apoA‑1; P<0.001). Furthermore, NBD‑cholesterol efflux in the THP‑1 cells exhibited a similar trend to that obseved in the PBMCs. In conclusion, the results of the present study suggested that fluorescent NBD‑cholesterol can be used as a sensitive and specific probe in cholesterol efflux assays in THP‑1‑derived macrophages.