The subunits of the hemocyanin from the tarantula, Eurypelma californicum, were isolated, following dissociation at pH 9.6, by a sequence of chromatographic and electrophoretic steps. Fraction 2 (containing two chains, a and c2) and the constituent polypeptide chains of the dimeric subunit 4D (b and c4) were resolved by anion exchange chromatography at pH 8.9 and 6.5, respectively. Since c2 and c4 have different electrophoretic mobilities in polyacrylamide gradient gels, the total number of different polypeptide chains is seven. The amino acid compositions of the seven chains are reported. There are major differences for at least half of the amino acids, while more consistent proportions become evident, if the amino acids are grouped by types of side chains. The N-terminal amino acid is proline in the case of chains b and e,, while no end group called be detected in any of the other chains by different methods. The C-terminal end group was found to be valine in both chains d and e. Cleavage by 70% formic acid, and by cyanogen bromide in formic acid results in fragmentation patterns distinct for each chain. After cyanogen bromide cleavage, the two largest peptides of each chain are of molecular weight near 2400. Tryptic fingerprints also reveal significant differences between all chains. Subunit heterogeneity of Eurypelma hemocyanin is clearly not the consequence of secondary modifications, but resides in major differences of the amino acid sequences.