Staphylococcus aureus is a common cause of prosthetic joint infection (PJI). The prevalence of superantigens (SAgs) among PJI-associated S. aureus is unknown. Eighty-four S. aureus isolates associated with PJI isolated between 1999 and 2006 were studied. SAg genes, sea , seb , sec , sed , see , seg , seh , sei , and tst , were assayed by PCR. Seventy-eight (92.9%) isolates carried at least 1 SAg gene studied, with 61 (72.6%) harboring more than 1. seg was most commonly (70.2%), and seh was least frequently (4.8%) detected. tst -positive isolates were associated with early infection and increased erythrocyte sedimentation rate at diagnosis ( P =0.006 and P =0.021, respectively). seg and sei were associated with methicillin resistance ( P =0.008 and P =0.002, respectively). A majority of PJI-associated isolates studied produced biologically active SAgs in both planktonic and biofilm growth modes. SAg genes are prevalent in S. aureus causing PJI.