Monoclonal antibodies (MAbs) against the rubella virion were used to locate epitopes involved in hemagglutination and neutralization. The MAbs exhibiting high-level hemagglutination-inhibiting activity were shown by Western blot analysis to be specific for the El polypeptide; this is consistent with the presence of the hemagglutinin on the El polypeptide. Some of the El-specific MAbs also neutralized viral infectivity. However, hemagglutination-inhibiting activity and neutralizing activity did not always correlate. Three distinct functional epitopes were identified on the El polypeptide by competition analyses: (i) one which reacted with MAbs with high-level hemagglutination-inhibiting activity and with neutralizing activity, (ii) one which reacted with MAbs with low-level hemagglutination-inhibiting activity and with neutralizing activity, and (iii) one which reacted with MAbs with only hemagglutination-inhibiting activity. A MAb specific for the E2 polypeptide exhibited neutralizing activity. This E2-specific MAb and two El-specific MAbs with neutralizing activity were capable of precipitating intact virus which indicates that at least three epitopes involved in neutralization are accessible on the surface of the virion.