Subfractionation of sarcoplasmic reticulum from fast‐twitch and slow‐twitch rabbit skeletal muscles was performed on a sucrose density gradient. Vesicle fractions were characterized by: measurement of (Ca²⁺,Mg²⁺)‐dependent (extra) ATPase, Mg²⁺ ‐dependent (basal) ATPase, Ca²⁺ uptake characteristics, polypeptide patterns in sodium dodecylsulphate polyacrylamide gel electro‐phoreses, phosphoprotein formation and electronmicroscopy of negatively stained samples.In fast‐twitch muscle, low and high density vesicles were separated. The latter showed high activity of (Ca²⁺, Mg²⁺)‐dependent ATPase, negligible activity of Mg²⁺‐dependent ATPase, high initial rate and high capacity of Ca²⁺ uptake, high amount of phosphorylated 115000‐M_r polypeptide, and appeared morphologically as thin‐walled vesicles covered with particles of 4 nm in diameter.Low density vesicles had little (Ca²⁺, Mg²⁺)‐dependent ATPase but high Mg²⁺‐dependent ATPase. Although the initial rate of Ca²⁺ uptake was markedly lower, the total capacity of uptake was comparable with that of high density vesicles. Phosphorylated 115000‐M_r polypeptide was detectable at low concentrations. Instead, 57000 and 47000‐M_r polypeptides were characterized as forming stable phosphoproteins in the presence of ATP and Mg²⁺. Negatively stained, these vesicles appeared to have smooth surfaces.It is suggested that low density vesicles represent a Ca²⁺ sequestering system different from that of high density vesiclés and that Mg²⁺‐dependent (basal) ATPase as well as the 57000 and 47000‐M_r polypeptides are part of the Ca²⁺ transport system within the low density vesicles. According to the results from slow‐twitch muscle, Ca²⁺ sequestration by the sarcoplasmic reticulum functions in this muscle type only through the low density vesicles.

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