The effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) on de novo purine synthesis was studied in rat liver in vivo and in primary cultured rat hepatocytes in vitro. In the in vivo system, malotilate increased [14C]glycine incorporation by 1.4- to 1.7-fold into acid soluble hepatic purines 4 to 18 hr and the rapidly miscible glycine pool size at least by 2.0-fold 12 hr after administration. Malotilate did not change 5-phosphoribosyl-1-pyrophosphate concentration but increased significantly amidophosphoribosyltransferase (E.C. 184.108.40.206) activity per unit of DNA or per unit of liver protein. Malotilate also increased significantly the hepatic cyclic AMP concentration 2 to 12 hr after administration. In the in vitro system, malotilate increased [14C] formate incorporation into acid soluble purines with accompanying increases in 5-phosphoribosyl-1-pyrophosphate availability and the specific activity of amidophosphoribosyltransferase. These results suggest that malotilate increases the rate of de novo purine synthesis in the liver by its direct action on hepatocytes, the mechanism of this increase is due to the combined increase of amidophosphoribosyltransferase activity and 5-phosphoribosyl-1-pyrophosphate supply and cyclic AMP may play a role for this response.