37S hemocyanin isolated from the tarantula Eurypelma californicum was subjected to a variety of fractionation procedures: Electrophoresis in polyacrylamide gels, isoelectrofocusing, and ion exchange chromatography. Single fractions were dissociated at alkaline pH and/or by sodium dodecyl sulfate and the resulting subunits separated by polyacrylamide gel electrophoresis in gradient slab gels followed, in many cases, by quantitative estimation of the subunit bands. In addition, crossed immunoelectrophoresis was employed to analyze the subunit composition. Separation of the native hemocyanin into fractions with different subunit composition was not observed. Also, hemocyanin samples taken from individual spiders showed a strikingly uniform composition. It is concluded that Eurypelma hemocyanin is homogeneous for all practical means. This implies that the seven different polypeptide chains described previously for Eurypelma hemocyanin all take part in the formation of the 24 subunits, 37S oligomeric molecule. By monitoring chromatographic effluents at 280 nm, scanning stained electrophoresis gels and determining peak areas in these experiments and in crossed immunoelectrophoresis, the relative quantities of the seven different polypeptide chains in the whole molecule were determined as follows (number per 37S molecule): 6a, 2b, 2c2, 2c4, 4d, 4e, 4f. The ratio between a and c2 (6:2) is the least well established. Since 7 different polypeptide chains are incorporated, the hexameric molecule cannot represent the basic oligomeric structure in tarantula hemocyanin, but probably the dodekamer. If Eurypelma hemocyanin is incubated with sucrose, partial dissociation occurs, yielding a fragment sedimenting with ca. 28S. This process is reversible. The 28S fragment still contains all subunit species; it is thought to represent a (3 x 6) or closely related structure. The implications of this finding with respect to the symmetry of the native hemocyanin molecule are discussed.