Hemagglutinin of vesicular stomatitis virus was prepared in suspension culture of BHK21/13 S cells maintained in a medium containing 0.4% bovine albumin and no serum. Optimal conditions for titration of VSV hemagglutinin included a low temperature, pH 5.8 and the use of goose erythrocytes. The hemagglutination pattern, once fully developed, remained fairly stable. Hemagglutinin was completely inactivated in one minute at 56°C; at 35°C it was stable for several days at pH 9.0. In density gradient centrifugation the peak HA activity was found in fractions of 1.22 to 1.24 g/ml, in which virions were also demonstrated by electron microscopy. Nonspecific inhibitors, present in high titers in normal rabbit serum, were removed by modified kaolin treatment. Specific HI titers in hyperimmune rabbit serum were low but increased slightly with ether- or fluoro-carbon-treated hemagglutinins and with a concentrated hemagglutinin.