Study of the human papillomavirus (HPV) E4 gene product has focused largely on HPV type 1 (HPV 1) primarily because of the large quantities of protein that can be purified from HPV 1-induced warts. We have extended the characterization of the HPV 1 E4 protein and, in this study, have shown that protein purified from clinical material and a heterologous expression system contains the divalent metal ion zinc. Furthermore, using a [65Zn]Cl2 dot-blot assay, we have shown that this binding is specific for zinc and those divalent cations that are known to structurally substitute zinc in metalloproteins. Mutational analysis has demonstrated that histidine amino acids (residues 56, 86, and 121), but not the cysteine residue (115), are essential for the zinc-binding activity of the E4 protein. Two assayable functions of E4 are dimerization and the formation of E4/cytokeratin structures in cultured cells; however, neither activity is abrogated by the loss of zinc binding.