The stress of routine transport practices on hatchery-reared brook troutSalvelinus fontinalis, lake troutSalvelinus namaycush, and splakeS. fontinalis×S. namaycushwas evaluated by measuring changes in plasma levels of the stress hormone cortisol, the key stress metabolite glucose, and the plasma electrolytes Na+and Cl–, and by measuring net ion and ammonia exchanges with the water in the transport tanks. We examined actual transport trips and standardized net confinement stress in the laboratory to quantify differences in stress response among the species. Brook trout were the least sensitive to both transport stress and net confinement, and responded in a similar fashion to both treatments. Splake responded to transport like brook trout but were the most sensitive to net confinement. Species differed most in their ability to maintain ion exchanges with the water under stress. Lake trout experienced ion losses during transport that were about 10-fold higher than losses from brook trout and splake. Trip duration (3.5–11 h) had only a minor effect on physiological responses, and wide variations in loading density (6.9–17 kg/100L) had no significant effect. Increasing O2levels in the water (due to oxygenation rather than aeration of the tanks) proved to be moderately stressful to brook trout, based on elevations in plasma cortisol levels. Our results permit an evaluation of the relative contributions of different transport variables to the transport stress imposed on salmonids.