Using antibodies specific for salmonid fish, we have determined the intracellular localization of hsp70, hsc70 and hsp90 before and after an acute heat shock in juvenile and mature rainbow trout. We found that both hsp70 and hsp90 were primarily located outside the nucleus in both the liver and the heart of juvenile and mature fish and heat shock resulted in an increase in these proteins in all cellular fractions examined. In mature fish, liver hsp70 was predominantly found in the membranes and organelles after heat shock, while in juvenile fish, hsp70 was mostly cytoplasmic. Hsc70 was found in all cellular compartments examined both before and after heat shock in the livers and hearts of juvenile and mature fish. Heat shock resulted in a significant induction of hsp90 in the liver tissue of both juvenile and mature fish; however, in juvenile fish, this increase was seen in the membranes and organelles whereas in mature fish, hsp90 increased in the nucleus and cytoplasm. Hsp90 was only induced in the hearts of mature fish with heat shock, where it increased in both the nucleus and the cytoplasm. These results indicate that the cells of juvenile and mature fish respond differently to acute temperature stress. While the nucleus appears to be an important target for hsp protection following heat shock, the presence of hsps in all subcellular fractions examined suggests multifunctional roles for these proteins in the cellular response to temperature stress in fish.