The three-dimensional structure of the cardiac muscle ryanodine receptor (RyR2) is described and compared with its skeletal muscle isoform (RyR1). Previously, structural studies of RyR2 have not been as informative as those for RyR1 because optimal conditions for electron microscopy, which require low levels of phospholipid, are destabilizing for RyR2. A simple procedure was devised for diluting RyR2 (in phospholipid-containing buffer) into a lipid-free buffer directly on the electron microscope grid, followed by freezing within a few seconds. Cryoelectron microscopy of RyR2 so prepared yielded images of sufficient quality for analysis by single particle image processing. Averaged projection images for RyR2, as well as for RyR1, prepared under the same conditions, were found to be nearly identical in overall dimensions and appearance at the resolution attained, '30