The HL-60 human leukemic promyelocyte can be induced to mature into terminally differentiated cells using certain nucleosides and chemotherapeutic agents. The mechanisms responsible for this induction of differentiation, however, remain unclear. We have monitored the effects of two specific inhibitors of DNA synthesis to determine whether slowing of DNA polymerization can induce HL-60 differentiation. The results demonstrate that cytosine arabinoside (ara-C) induces nonspecific esterase activity in HL-60 cells and increases surface expression of the monocyte antigen MY-4. The results also demonstrate that aphidicolin, an inhibitor of DNA polymerase which is not incorporated in DNA, induces similar phenotypic changes. The induction of differentiation by both agents was accompanied by loss of clonogenic potential as monitored by colony formation in methylcellulose. These observations suggest that terminal differentiation of HL-60 cells can be induced by drugs known to inhibit DNA synthesis.