We determined the effect of 1-hexylcarbamoyl-5-fluorouracil (HCFU), a masked compound of 5-fluorouracil, on the expression of heat shock protein (HSP) in heat-treated HeLa cells. We used a monoclonal anti-72-kDa heat shock protein antibody. At 37°C, HSP was predominant in the cytoplasm of cells and heating to 43 C for 30 min increased the synthesis of HSP in the nucleus. When HeLa cells were treated with heat and HCFU, at a concentration which showed evidence of synergy, nuclear staining of the cells decreased. Thus, the altered expression of HSP by HCFU is related to a synergism between heat and HCFU.