The phorbol esters induce differentiation of chronic lymphocytic leukemia (CLL) cells. Clinical use of this observation has been hampered by the fact that phorbol esters are also tumor promoters. In this study we demonstrate that another protein kinase C activator, without tumor promoting activity, has similar effects on CLL cells. Fresh leukemic cells from the peripheral blood of 13 patients with CLL were isolated and cultured in the absence (control) or presence of Bryostatin 1 or 12-0-tetradecanoylphorbol 13-acetate (TPA). Aliquots of cells were then analyzed after 24, 72 and 120 hours for morphological changes, acid phosphatase (ACP) and the co-expression of two hairy cell-associated surface antigens, CD22 and CD11c, by flow cytometry. Bryostatin 1 induced changes in shape and morphology similar to TPA, with adherence and increase in cell size, abundant cytoplasm and irregular cytoplasmic membrane. Both agents induced a statistically significant increase in the expression of CD22 and CD11c compared with control (p < 0.0008). There was no significant difference between the two agents in the degree of expression of these two markers. Both agents also induced ACP that was tartrate resistant (TRAP). These changes indicate that Bryostatin is as effective as TPA in inducing further differentiation of CLL cells to a hairy cell stage.