Previously [Inesi & de Meis (1989) J. Biol. Chem. 264, 5929-5936] it was shown that dimethyl sulphoxide (Me2SO) increases the amount of Ca2+ accumulated by sarcoplasmic-reticulum vesicles. This effect was related to a decrease in the enzyme affinity for ADP from less than 20 microM to 1 mM. In the present work the apparent affinity of the ADP-sensitive phosphoenzyme for ADP was determined by measuring the rate of ATP synthesis in vesicles previously loaded with Ca2+, at different pH values and in the presence and absence of Me2SO (20%, v/v) and KCl. In all conditions tested, addition of Me2SO never promoted an increase of the apparent Km for ADP to a value higher than 25 microM. ADP inhibits the phosphorylation of the enzyme by Pi. Two components, with Ki values of 80 microM and 8 mM, were detected when vesicles previously loaded with Ca2+ were used. The high-affinity component (Ki 80 microM) was abolished after addition of Me2SO to the medium. Empty vesicles, on the other hand, exhibited only the low-affinity component (Ki 8 mM). During ATP synthesis in a totally aqueous medium, there was a decrease in the phosphoenzyme formed by Pi, after addition of 80-100 microM-ADP to the medium. In the presence of Me2SO this decrease was smaller. The sum of the fractions of phosphoenzyme formed by ATP and by Pi during Ca2+ uptake was higher in the presence of Me2SO than in a totally aqueous medium. Me2SO decreased the passive efflux of Ca2+ measured in the presence of 0.1 mM-Pi and 0.1 mM-MgCl2. In a totally aqueous medium the same decrease was observed when Pi and MgCl2 concentrations were raised to 4 mM. These data suggest that ADP binds to two different enzyme conformations. The binding to one of these conformations (*E) inhibits the phosphorylation of the enzyme by Pi, increases the efflux of Ca2+ and decreases the amount of Ca2+ accumulated by the vesicles.