Embryonic stem (ES) cells were transfected with the protein-coding region of rat HSP27 cDNA placed in sense or antisense orientation in vector pcDNA3 under the control of the constitutive cytomegalovirus (CMV) promoter. Compared with nontransfected ES cells, five sense HSP27 cDNA-transfected ES cell clones displayed up to fourfold increases in expression of HSP27 mRNA and up to sixfold increases in expression of HSP27 protein, whereas four antisense HSP27 cDNA-transfected ES cell lines exhibited synthesis of antisense HSP27 RNA and a 50-85% decrease in HSP27 protein expression. Compared to the parental ES cell lines or ES cells transfected with the vector lacking any HSP27 sequence, all ES cell lines overexpressing HSP27 were resistant to killing by cadmium chloride (CdCl2), mercuric chloride (HgCl2), cis-platinum(II)-diammine dichloride (cDDP), sodium arsenite (NaAsO2), and heat while ES cell lines expressing reduced HSP27 were more sensitive to metal toxicity and heat. The relative toxicities of the tested metals to ES cells were cDDP > NaAsO2 > HgCl2 > CdCl2. Protection of ES cells against metal or heat toxicity was positively correlated with the level of HSP27. These data confirm in ES cells previous reports of the ability of HSP27 to protect other cell types against heat and demonstrate that HSP27 protects mammalian cells against the toxic effects of diverse metals.