Myocardial carnitine content of Fischer 344 rats decreases with age and proportionally affects total, free, acyl‐, and acetylcarnitine (ALC). In both adult and elderly hearts, ALC is the major acylcarnitine representing approximately 75% of the acylcarnitine fraction. The carnitine and ALC content of elderly hearts is restored by a single intraperitoneal (IP) injection of ALC (1.28mmol or 260mg/kg body weight) or by oral administration of ALC in drinking water (1.5%) over a two month period. In ALC‐treated rat hearts mitochondrial protein lysine acetylation is increased in both subsarcolemmal and interfibrillar mitochondria as assessed by immunoblotting. Moreover, there are qualitative differences in protein lysine acetylation between SSM and IFM. Furthermore, 2‐DIGE analysis showed that ALC induced changes in protein expression. Thirty one proteins showed a two‐fold or greater change, eight showed an increase and twenty three proteins a decrease. Identification of these proteins will direct our future studies into the mechanism of the physiological effects of acetylcarnitine.Supported in part by POI AG15885 and Sigma‐Tau SpA.