A systematic search for Escherichia coli proteins with the zinc-binding activity was performed using the assay of radioactive Zn(II) binding to total E. coli proteins fractionated by two methods of two-dimensional gel electrophoresis. A total of 30-40 radioactive spots were identified, of which 14 have been assigned from N-terminal sequencing. In addition to five known zinc-binding proteins, nine zinc-binding proteins were newly identified including: acetate kinase (AckA), DnaK, serine hydroxymethyltransferase (GlyA), transketolase isozymes (TktA/TktB), translation elongation factor Ts (Tsf), ribosomal proteins L2 (RplB), L13 (RplM) and one of S15 (RpsO), S16 (RpsP) or S17 (RpsQ). Together with about 20 known zinc-binding proteins, the total number of zinc-binding proteins in E. coli increased up to more than 30 species (or more than 3% of about 1000 proteins expressed under laboratory culture conditions). The specificity and affinity of zinc-binding were analysed for some of the zinc-binding proteins.