Purpose: Hyperthermia induces tumour cell apoptosis through the mitochondrial apoptotic pathway; however, the signal transduction mechanism underlying this process still needs to be fully elucidated. Phospholipid scramblase 3 (PLS3), a target of protein kinase C-delta (PKC-delta), resides in mitochondria and plays pivotal roles in regulating apoptotic response. Activated PLS3 facilitates cardiolipin (CL) translocation from the mitochondrial inner membrane to the outer leaflet of the mitochondrial outer membrane and triggers apoptosis. Materials and methods: The tongue squamous cell carcinoma Tca8113 cells were transfected or co-transfected using Lipofectamine 2000 with plasmids pCMV-6 x His-PLS3, pCMV-6 x His-PLS3 (T21A), pHA-PKC-delta, pHA-PKC-delta-KD (K376R), pHA-Hsp27, and empty control plasmid pcDNA3.1. The transfected cells were heated in water bath at 43 degrees C for 20 min, 40 min and 60 min. Assessments of apoptosis and redistribution of mitochondrial cardiolipin were performed by flow cytometry. PLS3, PKC-delta, Hsp27, phosphorylation of PLS3 and PLS3/PKC-delta interaction were detected by western blotting. Results: In our study the results show that elevated levels of the wild-type PLS3, but not the PLS3 (T21A) mutant, is able to increase hyperthermia-induced CL translocation and apoptosis. Wild-type PKC-delta facilitates PLS3 phosphorylation, PKC-delta/PLS3 interaction, and CL translocation, which consequently promote apoptosis. In contrast, heat shock protein 27 (Hsp27) blocks PKC-delta-induced PLS3 phosphorylation, suppresses PKC-delta/PLS3 interaction and CL translocation, and inhibits apoptosis. Conclusions: Our findings suggest that phosphorylation of PLS3 by PKC-delta is involved in the hyperthermia-induced apoptotic signal transduction pathway in Tca8113 cells, and that Hsp27 blocks this pathway to suppress hyperthermia-induced apoptosis.