A calcium binding characteristics of outside and inside vesicles as well as effects of K+, Mg2+, and acetylcholine cation on the calcium binding were studied in fragments of sarcoplasmic reticulum (SRF), isolated from frog femoral and abdominal muscles; high permeability of the preparations for Ca2+ was produced by incubation with I mM EDTA (pH 8-8.5) or with 5-10 microM ionophor X587A. Three types of Ca2+ binding were observed in the both SRF preparations: two of them exhibited the specific binding with high (K1) and low (K2) affinity and one type of unspecific binding with low affinity (Kunsp). The number of sites (n- nmol of Ca2+/mg of SRF protein) and dissociation constants (K microM) were the following SRF from abdominal muscle--n1 120, (K(1)15); n2 190, (K(2)135); Nunsp 650, (Kunsp 1625); SRF from femoral muscle--n(1)80, (K(1)64); n(2)265, (K(2)189); nunsp 500, (Kunsp 2240). The specific binding of Ca2+ in the SRF preparations was unaltered under physiological conditions in presence of KC1 0.1 M and MgCl2 1 mM if acetylcholine was added at concentration 10 mM, but the Ca2+ binding was completely inhibited at concentration 20 mM of acetylcholine. The unspecific Ca2+ binding was already inhibited at low concentrations of acetylcholine.