The influence of cell hydration and taurine on the heat shock response was studied in primary rat hepatocytes. Heat-induced accumulation of inducible heat shock protein 70 (HSP70) mRNA and protein was increased under hypo-osmotic conditions. In contrast, hyper-osmotic exposure blocked the HSP70 response during an 8-hour recovery, and this was paralleled by a reduction of overall protein synthesis and an impairment of thermotolerance. Taurine counteracted the hyper-osmotic inhibition of heat-induced HSP70 expression, but increased overall protein synthesis only slightly. A rapid and transient activation of the stress-activated protein kinase, JNK-2, was triggered by hyper-osmolarity, whereas the JNK-2 response to hypo-osmolarity was delayed. JNK-2 activation in response to heat was suppressed by hypo-osmolarity, but was markedly increased under hyper-osmotic conditions. The latter effect was blocked by taurine. A pronounced induction of the mRNA for the MAP-kinase phosphatase, MKP-1, in response to heat was observed during hypo- and normo-osmolarity, but no MKP-1 induction was found under hyper-osmotic conditions, although hyper-osmolarity itself led to accumulation of small levels of MKP-1 mRNA. Also, the block of heat-induced MKP-1 mRNA expression by hyper-osmolarity was abolished in the presence of taurine. The data provide evidence for a role of cellular hydration and taurine in the protection of liver parenchymal cells against heat injury via regulation of HSP70 expression and the balance between JNK-2 and MKP-1 activity.