Myosin V can be precipitated from brain soluble fraction under different assay conditions. In this work, myosin V from rat brain was precipitated by freezing of the soluble fraction. Brains were extracted and homogenized in 50 mM imidazole buffer, pH 8.0, containing 10 mM EDTA, 10 mM EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine.  The homogenate was centrifuged at 45.000 g for 40 min at 4°C and the supernatant stored at -20°C. Forty-eight hours later, the supernatant was thawed in a 27°C bath, and then centrifuged at 45.000 g for 40 min at 4°C. The precipitated fraction (P2) was homogenized in 20 mM imidazole buffer, pH 8.0, and again centrifuged at 45.000 g for 40 min at 4°C. SDS-PAGE analysis showed only 4 polypeptide bands in the precipitated fraction (P3): p200, p57, p45 and a band migrating at the front of the gel. The P3 fraction presented high Mg²⁺-ATPase activity (300 nmol/mg/min), which appeared to be associated with the high molecular weight polypeptide. This polypeptide was recognized by a specific myosin V antibody and was completely proteolized by calpain, generating two stable polypeptides: p130 and p90. The Mg²⁺-ATPase activity of the P3 fraction was slightly inhibited by 2 mM calcium in both the absence and presence of calmodulin. Although the K⁺/EDTA-ATPase activity of P3 represented only 20% of its Mg²⁺-ATPase activity, it was 3-fold higher than that of the control. This paper describes a new method for precipitating myosin V from brain supernatant, and the data suggest the presence of F-actin in this precipitate.