Dihydro-5-azacytidine (DHAC) is a hydrolytically stable congener of 5-azacytidine, which retains antileukemic activity against experimental leukemias. The biochemical pharmacology of DHAC was studied in tumor-bearing mice in order to elucidate the mode of action of this drug. We found that after an LD10 dose of DHAC, the plasma peak concentration achieved was 317 microM and was eliminated biexponentially, with a t1/2 alpha of 1.03 h and a t1/2 beta of 5 h. By 4 h, an unidentified metabolite of [3H]DHAC peaked and was eliminated biexponentially, with a t1/2 alpha of 1.06 h and a t1/2 beta of 10.6 h. [3H]DHACTP was the major anabolite in the L1210/0 cells, and was also eliminated biexponentially, with a t1/2 alpha of 4.3 h and a t1/2 beta of 12.2 h. An unknown anabolite of [3H]DHAC that eluted 5 min after [3H]DHACTP, between UTP and ATP, peaked at 3 h and could possibly be the deoxy-derivative [3H]DHAdCTP. A tissue distribution study revealed that the liver, L1210/0, and lung accumulate the most radioactivity per gram of wet tissue. Methylation studies showed that an LD10 dose of [3H]DHAC resulted in a 25.06% hypomethylation of DNA in L1210/0 cells and a 46.32% hypomethylation in a deoxycytidine kinase mutant cell line L1210/dCK(-), compared with their respective controls.