In this study, we explored the hypothesis that killifish acclimate to thermal extremes through regulation of genes involved in stress and metabolism. We examined the liver and gonadal transcription of heat shock proteins (hsp70, hsp90a, hsp90b), glucokinase (gck), and high mobility group b1 (hmgb1) protein in wild killifish species from hot springs and rivers using quantitative real-time PCR. Moreover, we exposed a river killifish species to a long-term thermal regime of hot spring (37-40 degrees C) and examined the liver transcription of the heat shock genes. Our results showed that hot spring killifish showed a significant, strong upregulation of liver hsp90a. Moreover, the testicular transcript levels of hsp90a, hsp90b, and hsp70 were higher in hot spring killifish than the river ones. The results of the common garden experiments showed that the transcripts of hsp70, hsp90b, and hmgb1 were mildly induced (> twofold) at the time when temperature reached to 37-40 degrees C, while the transcripts of hsp90a were strongly induced (17-fold increase). The level of hsp90a was dramatically more upregulated when fish were maintained in thermal extreme (42-fold change higher than in ambient temperature). Moreover, a significant downregulation of gck transcripts was observed at the time when temperature was raised to 37-40 degrees C (80-fold decrease) and during exposure to long-term thermal extreme (56-fold decrease). It can be concluded that the regulation of heat shock genes particularly hsp90a might be a key factor of the acclimation of fish to high temperature environments like hot springs. (c) 2015 Elsevier Inc. All rights reserved.