The expression of the molecular chaperone Hsp70 is induced upon heat shock and has critical roles in mediating proper protein folding. Heat shock induces a number of signal transduction pathways by modulating the activity of various cellular kinases and phosphatases. Suppression of these signalling pathways, in particular the c-jun N-terminal kinase (JNK) pathway, by Hsp70 is associated with inhibition of apoptosis. In this study, a human lymphoblastic T-cell line harbouring a tetracycline-regulated Hsp70 expression plasmid was used to examine the global changes in protein phosphorylation in heat-stressed cells and the influence of Hsp70 overexpression. Two-dimensional gel electrophoresis followed by phosphoprotein-specific fluorescent staining revealed a protein target observed to be highly phosphorylated in heat-stressed cells that had a markedly reduced level of phosphorylation in Hsp70-overexpressiog cells. The protein was identified by MALDI-TOF mass spectrometry as the actin-binding protein cofilin which depolymerizes filamentous actin in its unphosphorylated state. These findings indicate that heat shock induces the phosphorylation of cofilin thereby inactivating its depolymerizing activity. This may be an additional point at which Hsp70 overexpression increases apoptotic resistance.