The HL-60 human leukemic promyelocytic cell line can be induced to mature into terminally differentiated cells using certain chemotherapeutic agents. We have recently demonstrated that two inhibitors of DNA synthesis, cytosine arabinoside (ara-C) and aphidicolin, can induce HL-60 differentiation with the appearance of monocytic markers. These pyrimidine antimetabolites may have affected DNA methylation patterns and resulted in altered gene expression, or the differentiated phenotype may have occurred by inhibition of DNA replication. Consequently, we have extended these studies by using the purine analog, adenine arabinoside (ara-A), which also acts as an inhibitor of DNA synthesis. The results demonstrate that ara-A also induces HL-60 non-specific esterase activity and enhances expression of myeloid cell surface antigens, MY-4 and MO-1. The induction of a differentiated phenotype by ara-A occurs after partial inhibition of DNA synthesis, a finding similar to that observed with ara-C and aphidicolin. These observations indicate that purine, as well as pyrimidine analog inhibitors of DNA polymerization can induce differentiation of HL-60 cells along a monocytic lineage. These findings may be relevant to recent clinical trials that have employed low doses of ara-C in an attempt to induce differentiation of malignant hematopoietic cells.