Although the acute actions of short-acting β(2)-adrenoceptor agonists on force production in isolated mammalian skeletal muscle fibers have been the subject of a number of previous studies, those of long-acting β(2)-adrenoceptor agonists have never been investigated. Also, little is known about the cellular signal transduction events mediating their actions. Therefore, the primary aim of this study was to investigate the acute effects of treatment of mouse fast- and slow-twitch muscle fiber bundles with clenbuterol, formoterol, and salbutamol. Both clenbuterol and salbutamol increased the levels of cAMP in both fiber types, and this effect was reversed by ICI-118551. On the other hand, clenbuterol and formoterol decreased force production in both fiber types. They also increased the phosphorylation of phospholamban and β(2)-adrenoceptors in slow-twitch fiber bundles, and their effects were insensitive to propranolol, ICI-118551, and 14-22 amide. In contrast, salbutamol increased force production in both fiber types. It also increased the phosphorylation of β(2)-adrenoceptors in slow-twitch fibers only, but it had no effect on the phosphorylation of phospholamban in either fiber type. These effects were reversed by propranolol and ICI-118551 but not by 14-22 amide. Instead, 14-22 amide further potentiated the effects of salbutamol on force. In summary, long- and short-acting β(2)-adrenoceptor agonists have opposite effects on force production in isolated intact mouse skeletal muscle fiber bundles. From these results, we suggest that the acute actions of short-acting β(2)-adrenoceptor agonists on force production in mammalian skeletal muscles are mediated through the β(2)-adrenoceptor, whereas those of long-acting β(2)-adrenoceptor agonists are not.