Ubenimex (Bestatin), a low-molecular-mass dipeptide, has been demonstrated to have anti-tumor activities and immunomodulating activities. We here report cell growth inhibition and phenotypic changes of HL-60 and HL-60R cell lines induced by Bestatin treatment. Bestatin (0.1 microg/ml) showed remarkable cell growth inhibition against HL-60 cells, whereas it was ineffective for HL 60R cells. Bestatin also showed growth inhibition in the concentration of 1 microg/ml against HL-60R cells which are resistant to differentiation induction by DMSO and TPA. In both cell types, the effect of growth inhibition by Bestatin treatment was dose dependent. We found a low level expression of CD13 and a low number of CD13 positive cells in HL-60R cells compared with that of HL-60. We also observed phenotypic changes of HL-60 and HL-60R cells following incubation with Bestatin (10 microg/ml) for 1 and 3 hrs, respectively. With HL-60 cells, the upregulation of CD13/aminopeptidase N was found after 1 hr, however, the downregulation was observed after 3 hrs incubation with Bestatin. On the other hand, the downregulation of CD15 and CD33 was observed after both one and 3 hrs incubation. Similarly, in HL-60R cells, the upregulation of CD13/aminopeptidase N was found temporarily (1hr), and then CD13 downregulation was observed after 3 hrs incubation with Bestatin. No notable change was observed for expression of other myeloid-related antigens, e.g. CD14 (My4, LeuM3), CD11b (OKM1), and CD34 (My10). On the basis of these observations of in vitro activity, we suggest that Bestatin may also be an effective anti-leukemic agent in vivo.