We have previously developed a recombinant adenovirus containing a fusion gene of Escherichia coli cytosine deaminase ( CD ) and herpes simplex virus type 1 thymidine kinase ( HSV -1 TK ) controlled by a cytomegalovirus ( CMV ) enhancer -promoter. This replication -incompetent adenovirus effectively transduced the CD -TK gene into human prostate adenocarcinoma DU -145 or PC -3 cells. Interestingly, heat shock at 418C for 4 hours elevated the level of CD -TK by approximately 5 -to 20 -fold at a multiplicity of infection ( MOI ) of 1. Heat -enhanced expression of CD -TK promoted cytotoxicity by 23 -, 9 -, or 47 -fold in the presence of 50 g / mL ganciclovir ( GCV ), 500 g / mL 5 -fluorocytosine ( 5 -FC ), or 50 g / mL GCV + 500 g / mL 5 -FC, respectively, at an MOI of 1. Moreover, there was an increase in radiosensitivity when adenovirus -infected cells were heated at 418C for 4 hours followed by irradiation in the presence of the prodrugs. Virus + heat + 1 g / mL GCV treatment increased radiosensitivity by a dose -modifying factor ( DMF ) of 2.2, whereas virus + heat + 10 g / mL 5 -FC exposure resulted in a DMF of 2.3. Radiosensitization was clearly enhanced as a result of combined prodrug exposure ( DMF = 4.4 ). Our results suggest that the efficiency in expression of suicide genes from an adenoviral vector used for cytotoxic anticancer therapy could be improved by combining heat treatment with radiation therapy.