In human cells, alpha-lipoic acid (LA) is present in a bound lipoyllysine form in mitochondrial proteins that play a central role in oxidative metabolism. The possible effects of oral LA supplementation, a single bout of strenuous exercise and endurance exercise training on the lipoyllysine content in skeletal muscle and liver tissues of rat were examined. Incorporation of lipoyl moiety to tissue protein was not increased by enhanced abundance of LA in the diet. Endurance exercise training markedly increased lipoyllysine content in the liver at rest. A bout of exhaustive exercise also increased hepatic lipoyllysine content. A significant interaction of exhaustive exercise and training to increase tissue lipoyllysine content was evident. In vastus lateralis skeletal muscle, training did not influence tissue lipoyllysine content. A single bout of exhaustive exercise, however, clearly increased the level of lipoyllysine in the muscle. Comparison of tissue lipoyllysine data with that of free or loosely-bound LA results showed a clear lack of association between the two apparently related parameters. Tightly protein-bound lipoyllysine pool in tissues appeared to be independent of the loosely-bound or free LA status in the tissue.