In order to obtain a better understanding of the mechanism of the function of fragmented sarcoplasmic reticulum (FSR), we examined the binding of beta,gamma-methylene [3H]adenosine triphosphate (AMPOPCP), an unhydrolyzable ATP analogue, and 45Ca to FSR from bullfrog skeletal muscle. In medium containing 100 mM KCl and 20 mM Tris-maleate (pH 6.80) on ice, FSR has a single class of [3H]AMPOPCP-binding sites which amount to 4.4-8.6 nmol/mg protein (usually about 7 nmol/mg protein). The affinity was in the range of 6.2-12.3 X 10(3) M-1 in the absence of Ca2+. Ca2+ increased the affinity for AMPOPCP without changing the total number of binding sites, whereas Mg2+ decreased it. The change of the affinity is due to the direct effect of Ca2+ and Mg2+ on FSR. The possibility that Mg-AMPOPCP, Ca-AMPOPCP, and free AMPOPCP might have different affinities to FSR is excluded. The extent of Ca2+-induced enhancement in AMPOPCP binding is dependent not only on Ca2+ concentration but also on the concentration of AMPOPCP. The binding sites for AMPOPCP are likely to be the ATP-binding sites on Ca2+-ATPase protein on the basis of several lines of evidence, including competition between ATP, ADP, or AMP. FSR also binds 7-13 nmol Ca/mg protein (usually about 8 nmol/mg protein) with the affinity of 4-14 X 10(4) M-1 in the absence of the nucleotide in a similar medium containing 4 mM MgCl2. The ratio of Ca-binding sites to AMPOPCP-binding sites is mostly 1, but occasionally 2, corresponding to the ratio of Ca accumulated to ATP hydrolyzed by frog FSR. In the presence of a sufficient amount of the nucleotide, the affinity for Ca2+ was also increased. These findings are well explained by the random sequence binding model of Ca2+ and AMPOPCP, which bind to FSR with positive cooperative interaction between them. However, high concentrations of the nucleotide result in a negative cooperative interaction in the nucleotide binding in the presence of Ca2+, whereas no cooperativity is observed in the absence of Ca2+. Stimulation of Ca binding by AMPOPCP is also correspondingly affected. Comparative studies show that rabbit skeletal muscle FSR, in contrast to the frog one, shows negative cooperativity in its interactions with Ca2+ and AMPOPCP under some conditions and that the ratio of Ca-binding sites to AMPOPCP-binding sites is 2, corresponding to the well-known stoichiometry with ATP.