Bovine embryonic spleen cell cultures were examined to find several factors influencing the specificity, sensitivity and reproducibility of the syncytia infectivity assay of bovine leukemia virus (BLV). The highest sensitivity of the assay were observed when cell sheets of 30 to 50% confluence were inoculated with a stock of BLV, and when cells containing 4 or more nuclei were counted as syncytial cells. Treatment of the cell sheets with a diethylamino-ethyl-dextran solution (25 micrograms/ml) prior to BLV inoculation was found to be essential for the optimal induction of syncytia. Low-passage cultures were found to be more susceptible to the induction of syncytia by BLV than high-passage cultures. Cell-free BLV preparations decreased in syncytia-inducing ability to some extent by the first cycle of freezing (at -70 degrees C) and thawing. No further decrease, however, was caused by repeated cycles of freezing and thawing or by prolonged incuvation at -80 degrees C. The syncytia-inducing activity of BLV was inhibited by all the BLV-precipitating antibody-positive sera originated from both cases of the adult form of bovine leukosis and cases of persistent lymphocytosis. It was not inhibited by the sera of 16 of 17 cattle apparently healthy and negative for BLV-precipitating antibody. These results indicate that the syncytia infectivity assay and syncytia inhibition test are specific for BLV.