We have investigated the effects of water activity on oxygen-binding properties of giant invertebrate oxygen-binding proteins: hemocyanins from the intertidal crab, Carcinus maenas and the terrestrial snail, Arion ater and hemoglobin from the marine polychaete, Arenicola marina, using the osmotic stress method. We show that in contrast to the water-sensitive dimeric or tetrameric hemoglobins from humans, fish and cyclostomes, changes in water activity exert small effects on the oxygen affinity of these polymeric proteins, indicating that their deoxygenated and oxygenated states are almost similarly hydrated. The small effects of water activity may correlate with small surface to volume ratios in these high-molecular-weight proteins that pose a limit to water-accessible sites, and suggest smaller quaternary structural changes associated with oxygenation compared to those in dimeric and tetrameric hemoglobins.