Temporal patterns of biosynthesis of the sarcoplasmic reticulum protein, calsequestrin, were analyzed and compared with rates of ATPase synthesis in primary cultures of rat skeletal muscle cells. Rates of synthesis were measured by the incorporation of radioactive leucine into the isolated proteins. Cells at various stages of differentiation were incubated for 2 h with tritium-labeled leucine and extracted with detergent. The extracts were incubated with antibodies specific against calsequestrin or the ATPase and immunoprecipitates were separated by disc gel electrophoresis. Incorporation of radioactivity into bands identified as calsequestrin or the ATPase was analyzed by counting of gel slices. In Dulbecco's modified Eagles medium (DME medium) containing 0.1 volume of horse serum and 0.005 volume of chick embryo extract, the cells began to fuse after about 50 h in culture, forming multinucleated myotubes. Calsequestrin synthesis was barely detectable after 24 h in culture. After 44 h, before fusion of myoblasts began, the rate of calsequestrin synthesis increased severalfold. The rate of synthesis continued to increase until about 72 h and then diminished. If cells were transferred at 44 h to DME medium containing 0.2 volume of fetal calf serum and 0.08 volume of chick embryo extract, fusion was delayed by about 20 h. In this medium the rate of calsequestrin synthesis diminished after a peak at 44 h but, by contrast, the rate of synthesis of the ATPase increased dramatically following fusion at about 80 h. If cells were transferred at about 40 h to DME medium containing 0.1 volume of horse serum and only 60 muM Ca2+ the cells did not fuse and, again, the rate of calsequestrin synthesis was diminished after a peak at about 40 h. By contrast the rate of ATPase synthesis increased sharply in spite of the lack of fusion. Both proteins were degraded with a half-life of about 20 h. These studies show that the synthesis of calsequestrin, an extrinsic membrane protein, and the ATPase, an intrinsic protein of the same membrane, are synthesized under separate control.