Hagfish hemoglobin (Hb) is considered to represent a transition stage between invertebrate and vertebrate hemoglobins. The Hb system of Myxine glutinosa consists of three monomeric hemoglobins, which upon deoxygenation associate to form primarily heterodimers and heterotetramers. Myxine glutinosa is an osmoconformer, whose red blood cells show the exceptional ability to swell and remain swollen under hyposmotic conditions. In order to determine whether water activity regulates hemoglobin function, the effect of changes in osmolality on hemoglobin-O(2) affinity was investigated by applying the osmotic stress method to purified hemoglobins as well as intact red blood cells. Oxygen affinity decreases when water activity increases, indicating that water molecules stabilize the low-affinity, oligomeric state of the hemoglobin. This effect is opposite to that observed in tetrameric vertebrate hemoglobins, but resembles that seen in the dimeric hemoglobin of the marine clam Scapharca inaequivalvis. Our data show that water may act as an allosteric effector for hemoglobin within intact red cells and even in animals that do not experience large variations in blood osmolality.