A method has been developed to isolate cortical granules (CG) free in suspension. It involves the mechanical disruption of the CG from CG lawns (CGL; Dev. Biol. 43:62-74, 1975) and concentration of the CG by low speed centrifugation. The isolated CG are intact and are a relatively pure population as judged by electron microscopy. Granule integrity is confirmed by the fact that isolated intact CG are radioiodinated to only 0.05% of the specific activity of hypotonically lysed CG. Purity of the CG preparation is assessed by the enrichment (four- to sevenfold) of CG marker enzymes and the absence or low activity of plasma membrane, mitochondrial, cytoplasmic, and yolk platelet marker enzyme activities. CG isolated from 125I-surface-labeled eggs have a very low specific radioactivity, demonstrating that CG contamination by the plasma membrane-vitelline layer (PM-VL) is minimal. CG yield is approximately 1% of the starting egg protein. The CG isolation method is simple and rapid, 4 mg of CG protein being obtained in 1 h. Isolated CG and PM-VL display distinct electrophoretic patterns on SDS gels. Actin is localized to the PM-VL, and all bands present in the CGL are accounted for in the CG and PM-VL. Calmodulin is associated with the CGL, CG, and PM-VL fractions, but is not specifically enriched in these fractions as compared with whole egg homogenates. This method of isolating intact CG from unfertilized sea urchin eggs may be useful for exploring the mechanism of Ca2+-mediated CG exocytosis.