Prostaglandin D2 strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC50=2.09 x 10-5 M). Prostaglandin D2 also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D2-treated cells. Prostaglandin D2-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D2 pretreatment of the cells reduced endogenous DNA polymerase beta activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D2 had no direct inhibitory effect on the activity of either DNA polymerase alpha or beta. Our results indicate that the activities of DNA polymerase alpha and beta are lower in prostaglandin D2-treated mastocytoma cells. This finding accounts for the lower level of DNA synthesis in these cells.