The masses of inositol phosphates have been determined in isolated skeletal muscles from Xenopus laevis (sartorius, tibialis anterior and iliofibularis) and rat (gastrocnemius and soleus) which were quick-frozen in the resting state and at different stages of an isometric (Xenopus) or isotonic (rat) tetanus. The isomeric spectrum of inositol phosphates detected was similar to that in other tissues and cell types. The total sarcoplasmic concentrations of the isomers Ins-(1,4,5,6)P4/Ins(3,4,5,6)P4 (0.2-0.9 microM), Ins(1,3,4,6)P4 (not detectable), Ins(1,3,4,5,6)P5 (about 1 microM) and InsP6 (3.2-4.6 microM) were lower than in other cell types. Variations in these concentrations were due to the muscle type rather than to the donor species. The putative second messenger Ins(1,4,5)P3, as well as its dephosphorylation product Ins(1,4)P2, were present at surprisingly high total myoplasmic resting concentrations, ranging from 1.2 to 2.5 microM and 3.5 to 6.9 microM respectively. Upon tetanic stimulation these two inositol phosphates in particular exhibited significantly increased total sarcoplasmic concentrations, up to 4.2 microM and 11.3 microM respectively, with a time scale of seconds. From the initial rate of increase in the total sarcoplasmic concentrations of Ins(1,4,5)P3 and its rapidly formed metabolic products, a minimal phosphoinositidase C (PIC) activity in tetanically activated Xenopus skeletal muscle of about 1.7-2.6 microM/s can be estimated. This PIC activity observed in vivo seems to be far too low to account for a functional role for Ins(1,4,5)P3 as a chemical transmitter in the fast excitation-contraction coupling (ECC) process in skeletal muscle. The presence of Ins(1,3,4,5)P4 in all muscle types is indicative of a Ca(2+)-activated Ins(1,4,5)P3 3-kinase activity. The rapid transient increases in Ins(1,3,4)P3 and Ins(1,3)P2 in isometrically contracting Xenopus muscles suggest that corresponding Ins(1,3,4,5)P4 phosphatases are operating in skeletal muscle as well. In all muscles investigated except rat soleus, the fructose 1,6-bisphosphate [Fru(1,6)P2] concentration increased substantially during a tetanus, up to about 2 mM. This increase is correlated with a simultaneous decrease in phosphocreatine, whereas the energy charge of the muscles was essentially unaffected by the applied tetani. The time course of the rise in Fru(1,6)P2 was used to model changes in the free concentrations of high-affinity aldolase-binding inositol phosphates during the course of a tetanus. These calculations demonstrate that the free concentration of Ins(1,4,5)P3 and other aldolase-bound inositol phosphates can increase much faster and to a larger extent than the corresponding total concentrations as a result of their competitive displacement from aldolase-binding sites by the rapidly rising concentration of Fru(1,6)P2.