The mechanism by which the lipid peroxidation product 4-hydroxynonenal and several other homologous, yet non biogenic aldehydes inhibit proliferation of cultured Ehrlich ascites tumor cells has been studied. Incubation of cells (5 X 10(-4)/ml) in a minimum essential medium supplemented with 10 or 20 microM 4-hydroxynonenal reduces the 36-hr cell count to 65 and 30% of the control value. The reduced growth rate is most likely due to a blockage of the DNA synthesis. Cells labelled by a [3H]-thymidine pulse prior to exposure to 4-hydroxynonenal (20 microM, 8 hr) showed no change of the specific radioactivity of the DNA, indicating that no de novo synthesis occurred in the presence of the aldehyde. In the absence of the aldehyde the specific radioactivity of the DNA decreased by 25%. A 2-hr incubation in the presence of 10 or 20 microM of 4-hydroxynonenal reduced [3H]-thymidine incorporation into the HClO4 insoluble fraction to 85 and 50% of the controls, but had no effect of the [3H]-thymidine and 86Rb uptake. Moreover, examination of the cell cultures by the Trypan Blue exclusion technique revealed that 20 microM 4-hydroxynonenal does not cause cell death. The high reactivity of 4-hydroxynonenal towards sulfhydryl groups suggests that the aldehyde inhibits DNA synthesis by interacting with a functional SH group of DNA polymerase. The specific action on DNA synthesis is abolished at an aldehyde concentration of 50 microM, which leads to 30% (6 hr exposure) and 95% (36 hr exposure) of dead cells. The cytostatic index (CI), i.e. concentration at 50% Trypan Blue positive cells/concentration at 50% inhibition of cell growth deducted from the dose effect curves is 3.0 for 4-hydroxynonenal. The other homologous 4-hydroxyalkenals with chain length of 5, 6, 7, 8, 10 and 11 carbon atoms also inhibit cell growth. The CI varied from 1.20 to 1.94, indicating that these non biogenic 4-hydroxyalkenals have a distinctively lower specific effect on proliferation than the biogenic 4-hydroxynonenal. The Michael adducts of 4-hydroxynonenal with glutathione and cysteine were nearly one order of magnitude less toxic than the free aldehyde, the CI (2.41 cysteine adduct, 2.06 glutathione adduct), however, were not improved since the growth inhibitory action was also reduced.