1. In birds, prolonged cold exposure induces the development of a non-shivering thermogenesis (NST) of muscular origin that may result from an increase in ATP-dependent cycling of Ca2+ between the sarcoplasmic reticulum (SR) and the cytosol. 2. Because fatty acids are thought to play a significant role in NST, we investigated the effects of palmitic acid and related metabolites on skeletal SR Ca2+ uptake and release in ducklings. 3. Ca(2+)-ATPase activity, 45Ca2+ release and [3H]ryanodine-binding measurements indicated that palmitic acid was without effect on the Ca(2+)-ATPase and Ca2+ release channel. Palmitoyl carnitine and palmitoyl coenzyme A inhibited the Ca(2+)-ATPase at concentrations > 20 microM whereas both activated the Ca2+ release channel at concentrations < or = 20 microM in a dose-dependent manner. 4. Palmitoyl carnitine stimulated [3H]ryanodine binding to skeletal but not cardiac SR vesicles. Induction of 45Ca2+ release was observed with long-chain (C > or = 14) but not with short-chain acyl carnitines (C < or = 12). 5. Long-chain acyl carnitines accumulated significantly in duckling skeletal muscle during cold acclimation. Accordingly, these results suggest that long-chain acyl metabolites may modulate SR Ca2+ cycling and its associated thermogenesis in vivo.