Sarcoplasmic reticulum fragments capable of accumulating calcium were isolated from rat skeletal muscle by differential and sucrose gradient centrifugation. The ability of these fragments to accumulate calcium was impaired by adding 2,2-bis-(p-chlorophenyl)-1,1,1-trichloroethane (DDT) to the assay medium at concentrations of 0.06 to 6 muM. DDT (6 muM) caused a sharp lag in calcium uptake, with an 82% reduction in reaction rate 30 sec after calcium was added and a 62% reduction after one min. Basal ATPase activity of the microsomal fraction was inhibited by DDT but the calcium-stimulated increment of ATP hydrolysis was not. The findings show that DDT hinders calcium uptake by sarcoplasmic reticulum, but by some means other than inhibition of the calcium-stimulated ATPase. An apparent antagonism between DDT and ouabain or oligomycin was indicated. We propose that the presence of the lipid-soluble DDT molecule within the membrane of the sarcoplasmic reticulum interferes with the normal rapid uptake of calcium ions required for muscle relaxation, and that this interference may contribute to loss of muscle control in organisms poisoned by DDT.