Toxic shock syndrome toxin 1 (TSST-1) was partially purified from culture supernatants by SP-Sephadex C-25 ion-exchange chromatography and subsequent Sephadex G-75 gel filtration. This protein had an apparent molecular weight of 24,000 and an isoelectric point of 7.0. The NH2-amino acid sequence for the first 40 residues agreed completely with that predicted from the known TSST-1 genome. Ouchterlony immunodiffusion with monospecific rabbit antisera demonstrated a single line of identity with reference TSST-1 as well as with three preparations obtained from other investigators. When the purity of the different TSST-1 preparations was examined by Coomassie blue or silver staining after SDS-PAGE, only the major band at molecular weight 24,000 was apparent. However, multiple additional bands were seen in all preparations when visualized either by double staining with Coomassie blue stain followed by silver stain or by immunoblot using pooled human serum. Further purification of our preparation by reverse-phase high-performance liquid chromatography eliminated some, but not all, extraneous antigens. A final purification step by preparative SDS-PAGE resulted in an eluted protein that yielded only the 24-kDa TSST-1 band and a 48-kDa dimer by immunoblot. This material was endotoxin free (sensitivity limit, 10 pg/mL) and retained biologic activity for induction of cachectin and production of interleukin 1 by human monocytes. These data emphasize the need for stringent methods of assessment of purity in TSST-1 preparations.