The salinity tolerance of brook sticklebacks, Culaea inconstans, freshwater ninespine sticklebacks, Pungitius pungitius, and freshwater fourspine sticklebacks, Apeltes quadracus, was studied and compared with the tolerance of Pimephales promelas, Notemigonus crysoleucas, and Umbra limi by increasing the salinity in steps of 10% seawater (3.5‰) at regular intervals. Culaea had a significantly lower salinity tolerance than Pungitius and Apeltes but had a significantly higher salinity tolerance than Pimephales, Notemigonus, and Umbra. Culaea recovered when returned to fresh water after an abrupt transfer to 100% seawater for 1.75 h or less. In Culaea, temperature had an effect on salinity tolerance but neither light duration nor acclimation in 20% seawater could be shown to have any effect. Apeltes had a significantly higher salinity tolerance than Pungitius at 8 °C but not at 16 °C. At 16 °C most feeding and fanning activity ceased at 60%, 80%, and 110% seawater, in Culaea, Pungitius, and Apeltes, respectively. In the Gasterosteidae the order of decreasing salinity tolerance and increasing utilization of the freshwater habitat is as Follows: Spinachia, Apeltes, Gasterosteus, Pungitius, and Culaea.