The binding of 45Ca2+ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg2+ (no ATP) binding was half-maximal at 1.4-10(-6) M Ca2+ and the maximal amount bound was 1.6 mumol/g protein. At less than 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K' = 0.8 - (10(6) M-1) and the Hill coefficient was 1.0. In the absence of Mg2+, binding was half-maximal at 3 - 10(-7) M Ca2+ and the maximal amount bound was 2.9 mumol/g protein. The Scatchard plot indicated two classes of sites with K' values of about 2 - 10(7) and 2 - 10(6) M-1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of the Mg2+ binding to about half of the total Ca2+ binding sites is suppressed and there is a strong positive cooperativity involving half of the ramaining sites.