The majority of protozoan myosins I possess tail domains composed of three distinct and conserved regions of sequence, referred to as tail homology regions 1, 2 and 3 (TH.1, TH.2 and TH.3). While the N-terminal approximately half of the tail (corresponding to TH.1) has been implicated in membrane binding, all or some portion of the C-terminal approximately half of the tail (corresponding to TH.2 plus TH.3) has been implicated in binding to F-actin in a nucleotide-insensitive fashion. Here we show, using fusion proteins containing portions of the Dictyostelium myosin IC (myoC) tail domain and F-actin sedimentation assays, that the ability of the myoC tail to bind to actin resides entirely within the glycine- and proline-rich TH.2 domain. The src-like TH.3 domain does not bind to actin, nor does it augment the binding properties of the TH.2 domain. In addition to defining more precisely the location of the actin binding site in the tail domain of a protozoan myosin I, these results have implications for the function of the src-like TH.3 domain in myosins I and other proteins.