Toxic shock syndrome (TSS) is a multisystem illness caused mainly by Staphylococcus aureus producing TSS toxin-1 (TSST-1). A variant of TSST-1 has been isolated from ovine mastitis S. aureus. This toxin, TSST-ovine (TSST-O) is only weakly T cell mitogenic, is nonpyrogenic, does not enhance endotoxin shock, and does not cause TSS in the miniosmotic pump model. The sequence of the ovine gene (tstO) differs from the TSST-1 gene (tstH) by 14 nucleotides that change seven amino acids in the mature protein of which two are in the C-terminal half. A gene fusion containing half of both tstH and tstO was made and cloned into S. aureus. The fusion protein contained the two C-terminal amino acid differences that are in TSST-O at residues 132 and 140. The fusion protein was not T cell mitogenic and did not elicit TSS in two rabbit models. Additional experiments used mutagenesis to change the lysine residue at position 132 of TSST-O to glutamate (TSST-OK132E), as exists in TSST-1, and to change the lysine residue of the human-ovine fusion at position 132 to glutamate (TSST-11140T). Both mutants were pyrogenic, enhanced endotoxin shock, and caused TSS in the miniosmotic pump model. However, the proteins were only partially T cell mitogenic. The restoration of lethality of TSST-O and the human-ovine fusion by changing the lysine to glutamate, as exists in TSST-1, indicates that residue 132 is important in lethality. The failure to regenerate complete T cell mitogenicity of the same mutants indicates that residues 132 and 140 are important for that activity.