Electron probe analysis of ultrathin cryosections with high spatial resolution was used to determine in situ the concentrations of Ca2+ and Mg2+ bound in the absence of ATP to myofilaments in the I and A-bands of skinned frog skeletal muscle. At 2.2 x 10(-11) M Ca2+ and 2.7 x 10(-9) M Mg2+, the inexchangeably bound Mg2+ in the I-band was equivalent to the amount of divalent cations known to be inexchangeably bound to F-actin, while the Ca2+ bound to the I-band was not significantly above zero. The bound Mg2+ in the I-band was not exchangeable with Ca2+ even when the skinned fibres were exposed to 10 mM Ca2+ solution. These results clearly indicate that Mg2+, rather than Ca2+, is the divalent cation bound to F-actin in the thin filaments in situ. In the presence of 1 mM Mg2+, the exchangeable Ca2+ bound to the I-band was increased as a function of the free Ca2+, while that in the A-band was not significantly changed with [Ca2+] up to 2 x 10(-5) M, and increased to approximately 0.8 mol Ca2+ per mol myosin at 10(-4) M Ca2+. At a saturating free Ca2+ in Tris-Cl solution, the bound Ca2+ content (2-3 mol Ca2+ per mol troponin) of the nonoverlapping I-band was unexpectedly low; the replacement of Tris with Na+ enhanced Ca2+ binding to the level equivalent to 3-4 mol Ca2+ per mol troponin. The depressant effect of Tris on Ca2+ binding was greater in the absence of Mg2+. High concentrations of Tris also reduced the maximum tension induced by 10(-4) M Ca2+ buffered with 10 mM EGTA. At 1.3 x 10(-7) M Ca2+, thought to be close to the cytoplasmic free Ca2+ in resting muscle, the I-band bound a significant amount of Ca2+: equivalent to about 1 mol Ca2+ per mol troponin. In rabbit myofibrils there was a significant amount (approximately 1.5 mol/mol myosin) of Ca2+ bound by the A-band at a free Ca2+ of 10(-4) M.