Two class XIV unconventional myosins from Toxoplasma gondii, Myosin-A (TgM-A) and Myosin-C (TgM-C), were characterized in terms of their biochemical properties and their expression in quiescent and motile stages of the parasite life cycle. In cell fractionation studies, both myosins partitioned with the major organelle/cell membrane fraction, and extraction studies indicated that both were tightly associated with membrane domains as detergent was necessary for their solubilization. In addition, both TgM-A and TgM-C demonstrated a hallmark feature of myosins in their ability to bind actin in the absence but not the presence of ATP. In parasites residing within the host cell parasitophorous vacuole, TgM-A was detected by immunofluorescence microscopy as a bright spot near the apical pole of the parasite. This pattern underwent a subtle change as the parasites became motile, with TgM-A then localizing more intimately with the parasite cell membrane domain in apically disposed spots or patches, consistent with the role of this myosin in gliding motility. TgM-C showed a distinct localization to the juxtanuclear region towards the apical pole of the parasite, consistent with an association with the Golgi apparatus.