The mechanism of action of the synthetic estrogen diethylstilbestrol (DES) was investigated in murine L5178Y lymphoblasts. The dose-survival curve of cells treated with DES in serum-free medium for 1 hr was characterized by a prominent shoulder followed by a simple exponential decline; the Do, the dose of DES reducing cell survival to 1/e, was 1.52 nmoles/ml. DNA single-strand breaks, as measured by the alkaline elution method, were observed in DES-treated cells, and these followed a dose-response relationship after an apparent threshold of 10 microM DES was exceeded. Protein-associated strand breaks, which represent the increment in single-strand breaks that occurs by exposing drug-treated cells to proteinase K, were also noted. DNA double-strand breaks as measured by filter elution technology at pH 9.6 were observed and increased markedly to reach a level of approximately 9000 rad equivalents at a DES concentration of 20 microM. The measured ratio (mean +/- S.E.) of single- to double-strand breaks induced by DES in L5178Y limphoblasts was 0.09 +/- 0.035. A comparison of the ratio of single- to double-strand breaks induced by DES to that observed following radiation suggested that all of the single-strand breaks produced by DES could be attributed to double-strand breaks. The close correspondence of the dose-response curve for cytocidal activity of DES with that obtained for induction of DNA double-strand breaks suggested that such breaks may play an important role in the mechanism of cell kill by DES.