Eurythermal ectotherms commonly thrive in environments that expose them to large variations in temperature on daily and seasonal bases. The roles played by alterations in gene expression in enabling eurytherms to adjust to these two temporally distinct patterns of thermal stress are poorly understood. We used cDNA microarray analysis to examine changes in gene expression in a eurythermal fish, Austrofundulus limnaeus, subjected to long-term acclimation to constant temperatures of 20, 26 and 37 degrees C and to environmentally realistic daily fluctuations in temperature between 20 degrees C and 37 degrees C. Our data reveal major differences between the transcriptional responses in the liver made during acclimation to constant temperatures and in response to daily temperature fluctuations. Control of cell growth and proliferation appears to be an important part of the response to change in temperature, based on large-scale changes in mRNA transcript levels for several key regulators of these pathways. However, cell growth and proliferation appear to be regulated by different genes in constant versus fluctuating temperature regimes. The gene expression response of molecular chaperones is also different between constant and fluctuating temperatures. Small heat shock proteins appear to play an important role in response to fluctuating temperatures whereas larger molecular mass chaperones such as Hsp70 and Hsp90 respond more strongly to chronic high temperatures. A number of transcripts that encode for enzymes involved in the biosynthesis of nitrogen-containing organic osmolytes have gene expression patterns that indicate a possible role for these 'chemical chaperones' during acclimation to chronic high temperatures and daily temperature cycling. Genes important for the maintenance of membrane integrity are highly responsive to temperature change. Changes in fatty acid saturation may be important in long-term acclimation and in response to fluctuating temperatures; however cholesterol metabolism may be most critical for short-term acclimation to fluctuating temperatures. The variable effect of temperature on the expression of genes with daily rhythms of expression indicates that there is a complex interaction between the temperature cycle and daily rhythmicity in gene expression. A number of new hypotheses concerning temperature acclimation in fish have been generated as a result of this study. The most notable of these hypotheses is the possibility that the high mobility group b1 (HMGB1) protein, which plays key roles in the assembly of transcription initiation and enhanceosome complexes, may act as a compensatory modulator of transcription in response to temperature, and thus as a global gene expression temperature sensor. This study illustrates the utility of cDNA microarray approaches in both hypothesis-driven and 'discovery-based' investigations of environmental effects on organisms.