The in vitro inhibitory action and metabolism of 1-beta-D-arabinofuranosylcytosine (ara-C) on human myeloid (HL-60), B-lymphoid (RPMI-8392), and T-lymphoid (Molt-3) leukemic cells was compared. Ara-C produced greater inhibitory effects in Molt-3 cells than in either HL-60 or RPMI-8392 cells. At a 48 h exposure, ara-C was 7 and 10 times more cytotoxic to Molt-3 cells than to HL-60 and RPMI-8392 cells, respectively. The total ara-C uptake to nucleotides and the formation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) was about 5 times greater in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The incorporation of ara-C into DNA was also higher in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The mean intracellular half-life of ara-CTP was 31.7, 59.4, and 155 min for RPMI-8392, HL-60, and Molt-3 leukemic cells, respectively. The Km and Vmax values of ara-C for deoxycytidine kinase and the feedback inhibition of this enzyme by ara-CTP in the different leukemic cell lines could not explain the differences in metabolism of this analogue in these cells. These data indicate the increased sensitivity of T-lymphoid leukemic cells to ara-C than as compared with B-lymphoid and myeloid leukemic cells was due to an increased rate of formation and a longer half-life of ara-CTP in the T-cells.