1. Four species of Gammarus were studied: the fresh-water G. pulex, the brackish water G. duebeni, and two normally marine species G. locusta and obtusatus, the former of which has also been recorded from brackish water.
2. The relation between osmotic pressure and chloride of the blood and of the external medium, after sudden transfer to salinities which could be withstood for at least 24 hr., is shown in Fig. 1.
3. The changes in blood osmotic pressure are due to salt and not to water movements.
4. The marine species G. obtusatus and locusta maintain a very hypertonic blood in dilute sea water and can withstand 50% (270 mM.) and 25% (135 mM.) sea water respectively.
5. The brackish water G. duebeni has a tolerance range from pure sea water to water containing a trace of salt, but is not as well adapted to fresh water as G. pulex.
6. For a wide salinity tolerance range two mechanisms are necessary, (a) for regulating the blood concentration within certain limits, and (b) for maintaining a low intracellular concentration of certain ions (e.g. C1) in spite of changes in blood concentration. Defection of the latter mechanism can alone account for the inability of G. pulex to withstand direct transfer to more than about 40% sea water (115 mM.).
7. On the basis of this work and that of others on other animals the following hypothesis is suggested. Adaptation to fresh water has proceeded by two main stages: (a) Probably by active ion absorption, a high blood concentration is maintained (as in Eriocheir sinensis and Telphusa fluviatile) and is associated with a large blood/tissue C1 gradient. Such animals can still be transferred suddenly to a high concentration of sea water. (b) Evolution of the renal salt-reabsorption mechanism, and lowering of both blood concentration and blood/tissue C1 gradient to levels more easily maintained (as in G. pulex and most fresh-water animals). The consequent loss of power to maintain a large blood/tissue C1 gradient entails inability to withstand transfer to more than low concentrations of sea water, unless, as in certain species, a special mechanism is evolved for preventing the blood concentration from rising.