Recent observations indicated that carotenoids affected the proliferation and differentiation of certain cell lines. In the current experiments, β-carotene and lutein were tested for the induction of differentiation with HL-60 cells, a bipotent promyelocytic leukemia cell line. Cultures were incubated with lutein (0.0-10.0 μmol/l), β-carotene (0.010.0 μmol/l), and retinoic acid (0.0-1.0 μmol/l); retinoic acid incubations were positive controls for the differentiation of HL-60 cells into granulocytes. The carotenoid-incubated cultures developed significantly more (p ≤ 0.05) differentiated cells than control cultures (vehicle alone). Morphology of the carotenoid-incubated cells indicated differentiation along the granulocytic pathway. The percentage of differentiated cells increased significantly throughout a seven-day incubation period to 25% (lutein), 35% (β-carotene), and 75% (retinoic acid). A dose response was found for lutein (0.0-10.0 μmol/l), but not for β-carotene. Throughout the incubation period, the percentage of differentiated cells in negative control cultures did not change, remaining at the initial background level (≃5%). These results provide evidence for carotenoid-induced differentiation of cells. Induction of cell differentiation by a carotenoid without (lutein) and with (β-carotene) vitamin A activity suggested a vitamin A-independent mode of action for ca rotenoids in cell differentiation.