AbstractAn improved method has been devised for the purification of cytoplasmic dynein from sea urchin eggs (Strongylocentrotus droebachiensis and S purpuratus). This protocol introduces three changes over a previously published procedure (Hisanaga and Sakai: J Biochem 93:87, 1983)—the substitution of diethylaminoethyl (DEAE)‐cellulose for hydroxylapatite chromatography, the elimination of sucrose density gradient centrifugation, and the use of phosphoceliulose chromatography. These changes reduce the time and increase the efficiency of the purification procedure. The purified egg cytoplasmic dynein has enzymatic properties in common with axonemal dynein, including ionic specificity (Ca++ATPase/ Mg++ATPase = 0.8) and inhibition by sodium vanadate and erythro‐9‐2,3‐hydroxynonyl adenine (EHNA). As assayed by silver staining of polyacrylamide gels, the cytoplasmic dynein is composed of two high molecular weight polypeptides ( > 300 kilodaltons) that comigrate with flagellar dynein heavy chains, and lesser amounts of three lower molecular weight bands. None of these polypeptides appears to contain bound carbohydrate. The purification procedure can be modified slightly to allow the preparation of cytoplasmic dynein in only 2 days from as little as 3–5 ml of packed eggs, a 20‐fold reduction over the previous method. This more rapid and efficient method will facilitate the investigation of cytoplasmic dynein in other systems where starting material is limited, including tissue culture cells and nerve axoplasm.