The organic cosolvents propylene glycol (PG) and polyethylene glycol 400 (PEG 400) have previously been shown to differ in their potential to cause muscle damage following im injection. PG was found to be more myotoxic than PEG 400, with indirect implications of the role of cytosolic calcium in mediating this damage. In the present study, the direct effects of these cosolvents were investigated on the sarcoplasmic reticulum (SR), the major intracellular muscle membrane that mobilizes calcium. The passive permeability of isolated SR microsomal vesicles to calcium was not affected by 5.3 and 10.5% (v/v) PG and PEG 400. At 10.5% (v/v), a concentration of the organic cosolvent that would not be unexpected at the injection site, PEG 400 stimulated calcium uptake by 40 and 140% in longitudinal tubular-derived and terminal cisternal-derived vesicles, respectively, without significantly altering the ATP hydrolytic activity of the calcium pump. The calcium pumping efficiency (Ca2+/ATP coupling ratio) was therefore also enhanced. On the other hand, 10.5% (v/v) PG did not significantly alter either calcium uptake or ATPase activity of the pump. PG stimulated calcium efflux from only the terminal cisternae vesicles via a pathway indicative of the ryanodine-sensitive calcium channel, as demonstrated by inhibition of PG-induced efflux by millimolar Mg2+. These results are consistent with multiple interactions of cosolvents with proteins in the membrane bilayer, with the distinction that the two cosolvents differentially influence the calcium pump and release channel, particularly at the terminal cisternae, where there is rapid change of calcium level during excitation-contraction coupling. These data provide further evidence for the role of calcium in mediating organic cosolvent-induced muscle damage. In addition, they provide a possible explanation for the myoprotective effect of PEG 400 (compared to PG) as a result of increased myoplasmic calcium removal and reduced calcium release.