We examined the movement of [3H]palmitate across giant sarcolemmal vesicles prepared from red and white muscle of rainbow trout ( Oncorhynchus mykiss). Red and white muscle fatty acid carriers have similar affinities for palmitate (apparent Km= 26 ± 6 and 33 ± 8 nM, respectively); however, red muscle has a higher maximal uptake compared with white muscle ( Vmax= 476 ± 41 vs. 229 ± 23 pmol·mg protein-1·s-1, respectively). Phloretin (250 μM) inhibited palmitate influx in red and white muscle vesicles by ∼40%, HgCl2(2.5 mM) inhibited palmitate uptake by 20-30%, and the anion-exchange inhibitor DIDS (250 μM) inhibited palmitate influx in red and white muscle vesicles by ∼15 and 30%, respectively. Western blot analysis of red and white muscle vesicles did not detect a mammalian-type fatty acid transporter (FAT); however, preincubation of vesicles with sulfo- N-succinimidyloleate, a specific inhibitor of FAT in rats, reduced palmitate uptake in red and white muscle vesicles by ∼15 and 25%, respectively. A mammalian-type plasma membrane fatty acid-binding protein was identified in trout muscle using Western blotting, but the protein differed in size between red and white muscle. At low concentrations of free palmitate (2.5 nM), addition of high concentrations (111 μM total) of oleate (18:0) caused ∼50% reduction in palmitate uptake by red and white muscle vesicles, but high concentrations (100 μM) of octanoate (8:0) caused no inhibition of uptake. Five days of aerobic swimming at ∼2 body lengths/s and 9 days of chronic cortisol elevation in vivo, both of which stimulate lipid metabolism, had no effect on the rate of palmitate movement in red or white muscle vesicles.