4-Hydroxynonenal (HNE), produced during oxidative stress, has an antiproliferative/differentiative effect in several tumor cells. Recently, it has been observed that oxidative stress accelerates telomere loss. The length of telomeres depends on the telomerase activity, and the catalytic subunit of telomerase (hTERT) is strongly up-regulated in most human cancers and inhibited by differentiating agents. In this paper the inhibitory effect of HNE on telomerase activity and hTERT expression in three human leukemic cell lines (HL-60, U937, ML-1) is reported. To investigate the molecular mechanism involved in hTERT down-regulation by HNE, the expression of several transcription factors was also studied: in all these cell lines, c-Myc was inhibited, Mad-1 was up-regulated, and Sp-1 was not affected. Moreover, in p53 wild-type ML-1 cells, HNE up-regulated p53 expression. In HL-60 cells, DNA binding activity of c-Myc and Mad-1 to the E-box sequence of the hTERT promoter was inhibited and up-regulated, respectively. In summary, HNE inhibits telomerase activity via decreased hTERT promoter activity, by modulating c-Myc/Mad-1 transcription factor expression.