The accumulation of lipofuscin to cardiomyocytes is a classical parameter of aging and is believed to reflect oxidative stress. NF-kB transcription factor complex is one of the cellular sensors which responds to oxidative stress and regulates gene expression. Our purpose was to study whether aging affects the level and distribution of DNA binding activities of NF-kB transcription factors both in cardiac sarcoplasm and nuclear extracts. We used electrophoretic mobility shift assays (EMSA) to characterize the DNA binding activities of NF-kB and two other transcription factors. AP-1 and Sp-1, in the myocardium of 4 months and 24 months old male and female NMRI-mice. The protein levels of p50, p52, and p65 components of NF-kB-complex and an inhibitory IkB-alpha/MAD-3 were assayed with Western blots. Surprisingly, aging upregulated by 123% the nuclear NF-kB binding activity in the male and female mice. The sarcoplasmic NF-kB activity, activated by deoxycholate, did not show any change during aging. Aging-induced increase in nuclear NF-kB protein-DNA binding activity was observed both by gel retardation and UV-crosslinking assays. In immunoblotting, the level of p52 component but not those of p50 and p65 components of NF-kB-complex was slightly increased in nuclear fractions. Aging did not affect the sarcoplasmic levels of p50, p52, and p65 proteins. Supershift EMSA assays showed that the nuclear NF-kB complex contained p50, p52, and p65 components. The level of inhibitory IkB-alpha/MAD-3 protein was unaffected by aging both in nuclear and sarcoplasmic fractions. Aging down-regulated the nuclear Sp-1 binding activities but did not affect AP-1 binding activities. Statistically significant sex-related differences did not appear in the aging responses of transcription factors. These results indicate that NF-kB transcription factor pathway is activated during aging in cardiac muscle and could be the signaling route regulating gene expression. However, the activation mechanism of NF-kB during aging whether oxidative stress responsive or not in vivo needs further studies.