The human papillomavirus type 16 (HPV 16) E6 is a 151 amino acid protein containing four metal-binding motifs, Cys-X-X-Cys. We constructed and characterized three mutants with Gly substitutions for Cys within the motif; for Cys-66, for Cys-136, and for both, respectively. Zinc binding to bacterially expressed E6 was markedly reduced by the substitution for Cys-66, but DNA binding was unaffected by any of these mutations. Immunofluorescence staining showed that, whereas the E6 expressed in monkey COS-1 cells appeared mostly nuclear, the Cys-66 mutant appeared cytoplasmic. Subcellular fractionation followed by immunoprecipitation showed that the E6 in COS-1 cells was located in the membrane, nuclear, and nuclear-wash fractions, but not in the soluble cytoplasmic fraction, and that the nuclear Cys-66 protein was markedly reduced. The mutant proteins in COS-1 cells appeared to be less stable than the wild type, because the immunofluorescent cells were fewer and because the E6 bands in autoradiograms were less dense. The substitution mutants lost their capacity to enhance HPV 16 E7 transformation of rat 3Y1 cells. The data indicate that Cys-66 plays a crucial role for zinc binding and nuclear localization of E6 and that both Cys-66 and Cys-136 are required for a stable or functional structure of E6.