The hypothesis that 4-hydroxynonenal (HNE), a product of lipid peroxidation, might negatively affect cell proliferation, arose from the observation that lipid peroxidation is very low in tumors. In leukemic cells HNE inhibited cell growth and reduced c-myc and c-myb expression. HNE also induced differentiation in different leukemic cell lines. In HL-60 human leukemic cells, HNE induced the accumulation of cells in the G(0)/G(1) phase of the cell cycle accompanied by a decrease of cyclins D1, D2, and A. Moreover, HNE caused an increase in p21 expression. As cyclin D/CDK2 and cyclin A/CDK2 phosphorylate pRB, these findings suggested that pRb phosphorylation could be affected by HNE. Hypophosphorylated pRb binds and inactivates the E2F transcription factors. HNE induced the dephosphorylation of pRb and the increase in pRb/E2F1 complexes, whereas pRb/E2F4 complexes were reduced, because HNE downregulated E2F4 protein expression. The analysis of E2F binding to the P2 c-myc promoter revealed that HNE caused a decrease in "free" E2F, as well as an increase in pRb (and pRB family members) bound to E2F, with consequent repression of the transcription. In conclusion, HNE reduces E2F transcriptional activity by modifying a number of genes involved in regulation of the pRb/E2F pathway.