The degree to which laboratory derived measures of salinity tolerance reflect the field distributions of freshwater biota is uncertain. In this paper we compare laboratory-derived acute salinity tolerance (LC50 values) of freshwater macroinvertebrates (range 5.5-76 mS/cm) and fish (range 2.7-82 mS/cm) from southeastern Australia with the salinity from which they have been collected in the field. Only 4% of the macroinvertebrates were collected at salinity levels substantially higher than their 72-h LC50 obtained from directly transferring animals from low salinity water to the water they were tested (direct transfer LC50). This LC50 value was correlated with the maximum salinity at which a species had been collected. For common macroinvertebrates, the maximum field salinity was approximated by the direct transfer 72-h LC50. For adult freshwater fish, 21% of species were collected at salinities substantially greater than their acute direct transfer LC50 and there was a weak relationship between these two variables. Although there was a weak correlation between the direct transfer LC50 of early life stages of freshwater fish and the maximum field salinity, 58% of the field distribution were in higher than their LC 50 values. In contrast, LC50 determined from experiments that acclimated adult fish to higher salinity (slow acclimation) provided a better indication of the field distribution: with only one fish species (7%) being in conflict with their maximum field salinity and a strong positive relationship between these variables. This study shows that laboratory measures of acute salinity tolerance can reflect the maximum salinity that macroinvertebrate and fish species inhabit and are consistent with some anecdotal observations from other studies.