This paper compares wild-type and two mutant beta-actins, one in which Ser14 was replaced by a cysteine, and a second in which both Ser14 and Asp157 were exchanged (Ser14-->Cys and Ser14-->Cys, Asp157-->Ala, respectively). Both of these residues are part of invariant sequences in the loops, which bind the ATP phosphates, in the interdomain cleft of actin. The increased nucleotide exchange rate, and the decreased thermal stability and affinity for DNase I seen with the mutant actins indicated that the mutations disturbed the interdomain coupling. Despite this, the two mutant actins retained their ATPase activity. In fact, the mutated actins expressed a significant ATPase activity even in the presence of Ca2+ ions, conditions under which actin normally has a very low ATPase activity. In the presence of Mg2+ ions, the ATPase activity of actin was decreased slightly by the mutations. The mutant actins polymerized as the wild-type protein in the presence of Mg2+ ions, but slower than the wild-type in a K+/Ca2+ milieu. Profilin affected the lag phases and elongation rates during polymerization of the mutant and wild-type actins to the same extent, whereas at steady-state, the concentration of unpolymerized mutant actin appeared to be elevated. Decoration of mutant actin filaments with myosin subfragment 1 appeared to be normal, as did their movement in the low-load motility assay system. Our results show that Ser14 and Asp157 are key residues for interdomain communication, and that hydroxyl and carboxyl groups in positions 14 and 157, respectively, are not necessary for ATP hydrolysis in actin.