Heat shock (HS) induces a wide variety of biological processes, including inhibition of protein synthesis, elevated expression of heat shock proteins, induction of thermotolerance, and apoptotic cell death in a dose-dependent manner. We compared phosphorylated proteins in heat-shocked and thermotolerant cells using proteome analysis. After HS treatment of control RIF-1 and their thermotolerant derivatives, TR-RIF-1 cells, cellular proteins were separated by two-dimensional gel electrophoresis and the phosphorylated proteins were detected with the anti-phosphotyrosine antibodies. We found that 93 proteins showed significant changes in phosphorylation between control and thermotolerant cells as a function of recovery time after HS; we identified 81 of these proteins with peptide mass fingerprinting using MALDI-TOF MS after in-gel trypsin digestion. These phosphorylated proteins exhibit various cellular functions, including chaperones, ion channels, signaling molecules, in transcription and translation processes, in amino acid biosynthesis, oxidoreduction, energy metabolism, and cell motility or structure, suggesting that HS turns on the various signaling pathways by activating protein-tyrosine kinases (PTKs). Of these, 20 proteins were previously identified phosphorylated proteins and 64 were newly identified. These proteins can be grouped into three families: 1) proteins highly phosphorylated in TR-RIF-1 cells at basal level and phosphorylated more significantly by HS in RIF-1 than TR-RIF-1; 2) proteins highly phosphorylated in control RIF-1 cells at basal level and phosphorylated more easily by HS in TR-RIF-1 than in RIF-1 cells; and 3) proteins with a similar basal phosphorylation level in both RIF-1 and TR-RIF-I cells and responding to HS similarly in both cells. Most of the phosphorylated proteins are presumably involved in HS signaling in different ways, with the first and second families of proteins influencing thermotolerance. The possible tyrosine phosphorylation sites, the possible PTKs phosphorylating these proteins, and the proteins binding to these phosphorylated sites were predicted by the Netphos, ScanProsite, and Scansite programs. These results suggest that HS can activate various PTKs and HS responses can be regulated by phosphorylations of proteins having various functions.