A group of 2'-fluoro and 5-substituted arabinosyl pyrimidines and a group of base-substituted pseudoisocytidine analogs were evaluated for their capacity to induce differentiation in the human promyelocytic leukemia cell line, HL-60. These compounds were compared to 1-beta-D-arabinofuranosylcytosine (Ara-C) by monitoring: (1) inhibition of cell growth; (2) morphological maturation; (3) nitroblue tetrazolium (NBT) reduction; (4) expression of a myeloid differentiation antigen, Mo1; and (5) inhibition of colony formation. Exposure of logarithmically growing cells for 5 days to Ara-C, 2'-fluoro-Ara-C (FAC), 2'-fluoro-5-methyl-Ara-C (FMAC) and 2'-fluoro-5-ethyl-Ara-C (FEAC) resulted in cell growth inhibition at ED50 concentrations of 0.007, 0.11, 1.7 and 18 microM, and at cytostatic concentrations of 0.1, 0.5, 5.0 and 50 microM, respectively. These compounds induced granulocytic and monocytic maturation, reduction of NBT, increased expression of Mo1 antigen and a decrease or loss of both cell proliferation and colony formation in semisolid medium. There were few, if any, cell differentiation effects for the uracil nucleosides and pseudoisonucleosides tested. We found that Ara-C was the most cytotoxic of the compounds, and that when comparing absolute numbers of differentiated cells, i.e. percent of positive cells multiplied by the number of viable cells, FAC, FMAC and FEAC were superior to Ara-C inducing differentiation of HL-60 cells.