A method is described for the production of hybridoma cell lines secreting antibodies directed against the polyoma virus tumor antigens (T-Ags). Ten cloned lines have been established by this procedure and designated alpha Py Cl and C4, with activities against all three T-Ags; alpha Py LT1, -4 and -7, with activities against large T-Ag only; and alpha Py MT5, -7, -10, -13, and -16, with activities against middle T-Ag only. Some of the antibodies appear to react also with specific cellular protein. Data from studies on alpha Py MT7 point to the possible existence of more than one antigenic form of the viral middle T-Ag and, similarly, from studies on alpha Py LT7, to more than one form of large T-Ag. The approximate positions on the polyoma virus genome of the sequences encoding antigenic determinants have been mapped by using defined polyoma virus deletion mutants. The availability of these monoclonal antibodies allows the isolation of large amounts of viral T-Ags.