Understanding of the hydration state is an important issue in the chemomechanical energetics of versatile biological functions of polymerized actin (F-actin). In this study, hydration-state differences of F-actin by the bound divalent cations are revealed through precision microwave dielectric relaxation (DR) spectroscopy. G- and F-actin in Ca- and Mg-containing buffer solutions exhibit dual hydration components comprising restrained water with DR frequency f(2), (f(w)). The hydration state of F-actin is strongly dependent on the ionic composition. In every buffer tested, the HMW signal D-hyme (a (f(1)-f(w))delta(1)/(f(w)delta(w)) of F-actin is stronger than that of G-actin, where delta(w) is DR-amplitude of bulk solvent and delta(1) is that of HMW in a fixed-volume ellipsoid containing an F-actin and surrounding water in solution. Dhyme value of F-actin in Ca2.0-buffer (containing 2 mM Ca2+) is markedly higher than in Mg2.0-buffer (containing 2 mM Mg2+). Moreover, in the presence of 2 mM Mg2+, the hydration state of F-actin is changed by adding a small fraction of Ca2+ (similar to 0.1 mM) and becomes closer to that of the Ca-bound form in Ca2.0-buffer. This is consistent with the results of the partial specific volume and the Cotton effect around 290 nm in the CD spectra, indicating a change in the tertiary structure and less apparent change in the secondary structure of actin. The number of restrained water molecules per actin (N-2) is estimated to be 1600-2100 for Ca2.0- and F-buffer and similar to 2500 for Mg2.0-buffer at 10-15 degrees C. These numbers are comparable to those estimated from the available F-actin atomic structures as in the first water layer. The number of HMW molecules is roughly explained by the volume between the equipotential surface of kT/2e and the first water layer of the actin surface by solving the Poisson-Boltzmann equation using UCSF Chimera.