Heat shock response in cultured cells has been studied extensively; however few data are available on heat shock response in an intact organ of a living animal. In this study we analyzed the kinetics of expression of the heat shock protein 70 gene family (heat shock protein 70, heat shock cognate protein 73 and glucose-regulated protein 78) in the liver of the thermally stressed rat. New synthesis of heat shock protein 70 and heat shock cognate protein 73 was shown in liver slices pulse labeled in vitro with 35S-methionine. Accumulation of heat shock protein 70 and heat shock cognate protein 73 proteins was shown in total cellular extracts. 32P-labeled complementary DNA probes encoding heat shock protein 70, heat shock cognate protein 73 and glucose-regulated protein 78 were used to show that the levels of the corresponding messenger RNAs increase as a fraction of total RNA and in polysomes at different extents and with different kinetics. The induction of heat shock protein 70 and heat shock cognate protein 73 messenger RNAs reflected the increase in the synthesis of the corresponding proteins. Run-on transcription analysis indicated that the expression of heat shock protein 70 and heat shock cognate protein 73 genes was mainly regulated at the transcriptional level. On the contrary, both transcriptional and posttranscriptional regulatory mechanisms can explain the induction of the glucose-regulated protein 78 gene. The binding of a transiently activated heat shock factor to the heat shock element was detected by gel retardation assays and was directly correlated to the increase in the transcription of the heat shock protein 70 gene. In hyperthermic livers the increase in the expression of the heat shock protein 70 gene family was associated with an increase in the expression of the early response genes c-fos and c-jun. The observed differences in timing of expression and in regulatory mechanisms of different members of the heat shock protein 70 family are probably related to the specific liver response to an increase in body temperature. (Hepatology 1994;20:975–983).