Betulinic acid (BA) is a pentacyclic triterpene found in a number of medicinal plants and has been shown to cause apoptosis in a number of cell lines. We report here that BA may also have an effect on HL-60 cell differentiation. BA was cytotoxic to HL-60 cells with an IC50 of 5.7 μM after a 72-h treatment. Flow cytometry analysis showed that after exposure to 1–12 μM of BA for 72 h, approximately 10% of viable cells were in the sub-G1, presumably apoptotic, phase. At the same time differentiation was induced in approximately 10% (at 1 μM BA) to a maximum of 20% (at 6 μM BA) of cells as judged by the NBT-reduction test, and the expression of membrane markers CD11b and CD14. On the other hand, at 1 and 5 nM, 1α,25-dihydroxyvitamin D3 (DHD3) induced differentiation in approximately 10 and 70% of cells, respectively. At 1 nM DHD3, the addition of 1 μM BA increased differentiated cells from 10 to 43% and with 3 μM BA the increase was to 80%. BA also enhanced the effects of DHD3 in the expansion of the G1 cell population with a concomitant decrease of S phase cells. The effects of DHD3 and BA on CD11b and CD14 expression were inhibited by PD98059, a MEK inhibitor. Our results suggest that BA may enhance the effect of DHD3 in inducing mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase-mediated HL-60 cell differentiation.