Myosin I has been implicated as the motor that drives protrusion of the leading edge of motile cells. This function requires a close association with the plasma membrane and the cytoskeleton. Association with the actin cytoskeleton is mediated by an ATP-dependent binding site in the motor-containing myosin head, as well as by a second, ATP-independent actin binding site. In myosin IC from Acanthamoeba, the ATP-independent actin binding site is located in the carboxy-terminal tail, in a domain composed of two segments. The first segment is basic and is referred to as the GPA-rich segment. The second is a highly conserved sequence called src homology region 3 (SH3), found in a variety of cytoskeletal-associated proteins. We have used bacterially-expressed fusion proteins containing portions of Dictyostelium myosin IB to determine if the tail of this myosin I isoform also binds to actin and to establish precisely where the actin binding site is located. We have determined that the carboxy-terminal portion of the tail of Dictyostelium myosin IB can bind to actin in an ATP-independent manner and that the actin binding site is contained within residues 922-1059, corresponding to the GPA-rich segment of Acanthamoeba myosin IC. We conclude that this region contains a specific actin binding site which may be responsible for the cytoskeletal association of this myosin I isoform.