DNA polymerase alpha from the leukemic cells of acute lymphoblastic leukemia (ALL) was found to be more resistant to the inhibition by 1-beta-D-arabinofuranosylcytosine 5'-triphosphate than that from acute myeloblastic leukemia (AML). Apparent Ki values for 1-beta-D-arabinofuranosylcytosine 5'-triphosphate of DNA polymerase alpha from eight patients with ALL [26.7 +/- 7.1 (S.D.) microM] were 5 times higher than those from nine patients with AML [5.2 +/- 1.3 microM]. In contrast, apparent Km values for a normal substrate deoxycytidine 5'-triphosphate of DNA polymerase alpha preparations from either AML and ALL were almost identical (9.4 to 10.9 microM). Likewise, apparent Ki values for another arabinoside analog, 9-beta-D-arabinofuranosyladenine 5'-triphosphate, of DNA polymerase alpha from blasts of seven patients with ALL (16.9 +/- 6.9 microM) were significantly higher than those from patients with AML (3.8 +/- 0.5 microM). These results indicate that DNA polymerase alpha from ALL blast cells has a decreased affinity to the arabinoside analogs of deoxynucleotide triphosphate. The sensitivity of DNA polymerase alpha of blast cells to 1-beta-D-arabinofuranosylcytosine 5'-triphosphate may be one of the determinants of the clinical response to 1-beta-D-arabinofuranosylcytosine treatment.