The oxygen-avid, perienteric hemoglobin of Ascaris is a homooctamer. Each subunit contains two tandem globin domains that are highly homologous with the exception of a charged COOH-terminal extension. In solution, recombinant domain one (D1) exists as a monomer, whereas recombinant domain two with the COOH-terminal tail (D2) is primarily an octamer. To examine the role of the COOH-terminal extension in Ascaris hemoglobin multimer formation, we attached the tail to the monomeric, heme-containing proteins, myoglobin and D1; neither construct was capable of multimer formation. Additionally, we removed the tail from both full-length Ascaris hemoglobin and D2. This substantially decreased, but did not eliminate, multimerization. We further characterized subunit interactions by disrupting full-length hemoglobin multimers with the chaotropic salt, NaSCN, which yielded intermediate oligomers. In solution, D2 demonstrated a greater propensity to dissociate than full-length hemoglobin, indicating that D1 contributes to octamer stability. D1 formed a weak dimer in its crystal; thus, we analyzed interactions along the subunit interface. Hydrogen bonds as well as hydrophobic and electrostatic forces appeared to contribute to dimer formation. Amino acid substitutions along this interface in D2 are predicted to enhance subunit interactions for that domain. Our studies reveal that the COOH-terminal tail is necessary, but not sufficient, for efficient octamer formation. Other regions, possibly within the E- and F- helices and AB loops of both domains, appear to be important for Ascaris hemoglobin octamer formation.