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Populations of common killifish, Fundulus heteroclitus, are distributed along the Atlantic coast of North America through a steep latitudinal thermal gradient. We examined intraspecific variation in whole-animal thermal tolerance and its relationship to the heat shock response in killifish from the northern and southern extremes of the species range. Critical thermal maxima were significantly higher in southern than in northern fish by ∼1.5°C at a wide range of acclimation temperatures (from 2–34°C), and critical thermal minima differed by ∼1.5°C at acclimation temperatures above 22°C, converging on the freezing point of brackish water at lower acclimation temperatures. To determine whether these differences in whole-organism thermal tolerance were reflected in differences in either the sequence or regulation of the heat shock protein genes (hsps) we obtained complete cDNA sequences for hsc70, hsp70-1 and hsp70-2, and partial sequences of hsp90α and hsp90β. There were no fixed differences in amino acid sequence between populations in either hsp70-1 or hsp70-2, and only a single conservative substitution between populations in hsc70. By contrast, there were significant differences between populations in the expression of many, but not all, of these genes. Both northern and southern killifish significantly increased hsp70-2 levels above control values (Ton) at a heat shock temperature of 33°C, but the magnitude of this induction was greater in northern fish, suggesting that northern fish may be more susceptible to thermal damage than are southern fish. In contrast, hsp70-1 mRNA levels increased gradually and to the same extent in response to heat shock in both populations. Hsc70 mRNA levels were significantly elevated by heat shock in southern fish, but not in northern fish. Similarly, the more thermotolerant southern killifish had a Ton for hsp90α of 30°C, 2°C lower than that of northern fish. This observation combined with the ability of southern killifish to upregulate hsc70 in response to heat shock suggests a possible role for these hsps in whole-organism differences in thermal tolerance. These data highlight the importance of considering the complexity of the heat shock response across multiple isoforms when attempting to make linkages to whole-organism traits such as thermal tolerance. [ABSTRACT FROM AUTHOR]