We purified Myr3 (third unconventional myosin from rat), a mammalian "amoeboid" subclass myosin I, from rat liver. The heavy chain of purified Myr3 is associated with a single calmodulin light chain. Myr3 exhibits K/EDTA-ATPase and Mg-ATPase activity. The Mg-ATPase activity is stimulated by increasing F-actin concentrations in a complex triphasic manner similar to the Mg-ATPase activity of myosin I molecules from protozoa. Although purified Myr3 was observed to cross-link actin filaments, it bound in an ATP regulated manner to F-actin, and no evidence for a nucleotide-independent high affinity actin binding site that could explain the triphasic activation pattern was obtained. Micromolar concentrations of free Ca2+ reversibly inhibit the Mg-ATPase activity of Myr3 by binding to its light chain calmodulin, which remains bound to the Myr3 heavy chain irrespective of the free Ca2+ concentration. Polyclonal antibodies and Fab fragments directed against the tail domain were found to stimulate the Mg-ATPase activity. A similar stimulation of the Myr3 Mg-ATPase activity is observed upon proteolytic removal of the very C-terminal SH3 domain. These results demonstrate that Myr3 is subject to negative regulation by free calcium and its own tail domain and possibly positive regulation by a tail-domain binding partner.