The number and size of the major virion polypeptides have been determined by SDS-PAGE for the 22 Staphylococcus aureus phages of the International Typing Set, plus phages 11 and 80 alpha. Virion ultrastructure was examined by electron microscopy after negative staining with ammonium molybdate. In addition, serogroup B phages were disrupted and fractionated into head, tail-tube and baseplate components and major polypeptides assigned to these substructures. The number and size of the polypeptides correlated closely with the division of aureophages into four serogroups (A, B, F, L), although serogroup L was represented in the set by only a single phage (187). Apart from serogroup B, however, the polypeptide patterns did not reflect differences between lytic groups. Within serogroup B, polypeptide analysis yielded characteristic patterns for lysogroups I, II and III. Ultrastructural analyses confirm the data provided by polypeptide analysis. Thus, phages from the four serogroups can be identified on the basis of tail-tube length alone, although the differences between phage 187 and members of lysogroups I and III in serogroup B were less than 20 nm, or approximately 12% of the total length. Serogroup A virions differed from those of the other serogroups in that all members of the typing set in this group had elongate, rather than isometric, heads.