Actin can be specifically cleaved between residues 42 and 43 with a novel protease from Escherichia coli A2 strain (ECP) [Khaitlina, S. Y., Collins, J. H., Kuznetsova, I.M., Pershina, V.P., Synakevich, I.G., Turoverov, K.K. & Usmanova, A.M. (1991) FEBS Lett. 279, 49-51]. The resulting C-terminal and N-terminal fragments remained associated to one another in the presence of either Ca2+ or Mg2+. The protease-treated actin was, however, neither able to spontaneously assemble into filaments nor to copolymerize with intact actin unless its tightly bound Ca2+ was replaced with Mg2+. Substitution of Mg2+ for the bound Ca2+ was also necessary to partially restore the ability of the protease-treated actin to inhibit the DNase I activity. The critical concentration for KCl-induced polymerization of ECP-treated ATP-Mg-G-actin, determined by measuring the fluorescence of pyrenyl label, was approximately threefold higher than that for actin cleaved between residues 47 and 48 using subtilisin, and 36-fold higher than the critical concentration for polymerization of intact actin under the same conditions. Morphologically, the filaments of ECP-treated actin were indistinguishable from those of intact actin. Comparison of the fluorescence spectra of pyrenyl-labelled actins and chemical cross-linking with N,N'-1,2-phenylenebismaleimide have, however, revealed structural differences between the filaments assembled from ECP-treated actin and those of intact as well as subtilisin-treated actin. Moreover, the filaments of ECP-treated actin were easily disrupted by centrifugal forces or shearing stress unless they were stabilized by phalloidin. The results are consistent with the direct participation of the region around residues 42 and 43 in the monomer/monomer interactions as predicted from the atomic model of F-actin [Holmes, K.C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44-49] and suggest that the interactions involving this region are of primary importance for stabilization of the actin filament. The mechanism of the regulation of actin polymerization by the tightly bound divalent cation is also discussed.