To assess more systematically functional differences among non-muscle and muscle actins and the effect of specific mutations on their function, we compared actin from Dictyostelium discoideum (D-actin) with actin from rabbit skeletal muscle (R-actin) with respect to the formation of filaments, their three-dimensional structure and mechanical properties. With Mg(2+) occupying the single high-affinity divalent cation-binding site, the course of polymerization is very similar for the two types of actin. In contrast, when Ca(2+ )is bound, D-actin exhibits a significantly longer lag phase at the onset of polymerization than R-actin. Crossover spacing and helical screw angle of negatively stained filaments are similar for D and R-F-actin filaments, irrespective of the tightly bound divalent cation. However, three-dimensional helical reconstructions reveal that the intersubunit contacts along the two long-pitch helical strands of D-(Ca)F-actin filaments are more tenuous compared to those in R-(Ca)F-actin filaments. D-(Mg)F-actin filaments on the other hand exhibit more massive contacts between the two long-pitch helical strands than R-(Mg)F-actin filaments. Moreover, in contrast to the structure of R-F-actin filaments which is not significantly modulated by the divalent cation, the intersubunit contacts both along and between the two long-pitch helical strands are weaker in D-(Ca)F-actin compared to D-(Mg)F-actin filaments. Consistent with these structural differences, D-(Ca)F-actin filaments were significantly more flexible than D-(Mg)F-actin. Taken together, this work documents that despite being highly conserved, muscle and non-muscle actins exhibit subtle differences in terms of their polymerization behavior, and the three-dimensional structure and mechanical properties of their F-actin filaments which, in turn, may account for their functional diversity.