Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] represents a useful approach for the treatment for cancer, including acute myeloid leukemia (AML). Recent studies demonstrated that the combined administration of 1,25-(OH)(2)D(3) and differentiation-enhancing agents could alleviate the side effects of 1,25-(OH)(2)D(3) and improve the rate of long term survival. In this study, we determined the enhancing activities of ceramide derivatives on 1,25-(OH)(2)D(3)-induced differentiation of human myeloid leukemia HL-60 cells. Importantly, some of these derivatives -- namely, A2, B3, and H9 -- enhanced the 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells in a concentration-dependent manner. In addition, the morphologic studies using Giemsa staining and flow cytometric analysis demonstrated that the combined treatment of 1,25-(OH)(2)D(3) with one of the three analogues, A2, B3, and H9, directed the HL-60 cells into monocytic lineage, but not into granulocytic lineage. The inhibition studies demonstrated that A2, B3, and H9, enhanced 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells via the PI3-K/PKC/JNK/ERK pathways. The ability of ceramide derivatives to enhance the differentiation-inducing potential of 1,25-(OH)(2)D(3) may contribute to an effective therapy for AML.