G-actin has a single tight-binding (high-affinity) site for divalent cations per mole of protein, whose occupancy is important for the stability of the molecule. Different tightly bound divalent cations differently influence the polymerization properties of actin. The tightly bound metal ion easily exchanges for free exogenous cations. Moreover, biochemical and structural evidence demonstrates that actin, in both the G- and F-forms, assumes different conformations depending on the metal ion bound with high affinity in the cleft between two main domains of the molecule. In this work, we used proteolytic susceptibility to detect possible local conformational alterations of the actin molecule following a brief incubation of Ca-G-actin with barium chloride and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We found that substitution of Ba2+ for the tightly bound Ca2+ affects the regions around Arg-62 and Lys-68 in subdomain 2 of G-actin, as judged from inhibition of tryptic cleavage at these residues. Using the fluorescent chelator Quin-2, we observed that about 0.95 mol of Ba2+ is released per 1 mol of actin. We also examined the effect of replacement of the tightly bound Ca2+ by Ba2+ on actin polymerization. With respect to Ca-actin, Ba-actin shows an increased polymerization rate, mainly due to its enhanced nucleation and a higher critical concentration.