We developed a reliable method for transient transfection of Acanthamoeba using Superfect (Qiagen) and a vector with the Acanthamoeba ubiquitin promoter and enhanced green fluorescent protein (EGFP) as the reporter gene. The transfection efficiency was 3% for profilin-I-EGFP and EGFP-myosin-II tail, and less than 0.5% for larger constructs such as full length myosin-II or myosin-IC. Profilin-I-EGFP was distributed throughout the cytoplasm as observed previously with rhodamine-labeled profilin, while EGFP alone accumulated in the nucleus. EGFP fused to full length myosin-II or to the C-terminal 256 residues of the myosin-II tail concentrated in fluorescent spots similar to thick filaments and minifilaments identified previously in fixed cells with fluorescent antibodies. Thick filaments were located in the dorsal cytoplasm and along the lateral margins of the back half of the cell. Thick filaments formed behind the leading edge and moved continuously towards the rear of the cell, where they disassembled. If phosphorylation of the myosin-II heavy chain was prevented by mutation of all three phosphorylated serines to alanine, thick filaments of unphosphorylated myosin-II accumulated around vesicles of various sizes. EGFP-myosin-IC was spread throughout the cytoplasm but concentrated transiently around contractile vacuoles and macropinocytosis cups providing that the construct included both the head and a tail with the SH3 domain.