In this study, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) formation, retention, and incorporation into DNA were simultaneously evaluated in vivo in mice bearing leukemia cells sensitive to 1-beta-D-arabinofuranosylcytosine (ara-C) (L1210/0), leukemia cells resistant to ara-C (L1210/R), P288, and lymphosarcoma P1798, namely cells characterized by differential sensitivity to ara-C. In L1210/R cells, resistance to ara-C was correlated with low deoxycytidine-cytidine kinase activity (0.04 nmol/mg protein/min), with a low level of intracellular accumulation of ara-CTP, with a low level of incorporation of ara-C into DNA, and with no significant inhibition of thymidine incorporation into DNA. Thus a simple measurement of the intracellular pool of total ara-C nucleotides is sufficient to identify cells with this type of resistance. In contrast, in cells with sufficient deoxycytidine-cytidine kinase activity (greater than 0.1 nmol/mg protein/min), the factors determining the quality of response to ara-C could be distinguished as follows: (a) those which are responsible for in vitro cytotoxicity (producing in vivo cytoreduction); and (b) those which are responsible for in vivo selectivity (producing long term survivors). In P288 cells which are sensitive in vitro to ara-C, the determining factor for this sensitivity is the amount of ara-CTP formed which produced greater than 80% inhibition of thymidine incorporation into DNA. The lack of antitumor activity in vivo, however, was due to similarities in ara-CTP retention in target tumor cells (P288) and normal bone marrow cells. In both cases, ara-CTP retention at 4 h was less than 10% of the value obtained at 30 min. In contrast, in cells such as L1210 and P1798 long term survivors (cures) were directly correlated with higher ara-CTP retention. For example, 4 h after drug administration, ara-CTP retentions were 20, 82, and 6% for L1210, P1798, and bone marrow cells, respectively. At 24 h, 20% ara-CTP was retained intracellularly by P1798 tumor cells. In summary, results presented herein demonstrate the importance of differential ara-CTP retention as the most critical determinant of response for the induction of long term survivors, and ara-C incorporation into DNA by tumor cells after in vivo treatment appears to be less significant. These data also demonstrate close correlation between ara-CTP pools, retention, and the extent of inhibition of recovery of thymidine incorporation into DNA.