Mouse mammary tumor viruses express a superantigen essential for their life cycle. It has been proposed that viral superantigens (vSags) require processing by prohormone convertases (PCs) for activity. We now observe, using a panel of mutant forms of potential PC cleavage sites and in vitro cleavage assays, that only the CS1 (position 68 to 71) and CS2 (position 169 to 172) sites are utilized by furin and PC5. Other members of the convertase family that are expressed in lymphocytes are not endowed with this activity. Furthermore, mutant forms of two different viral superantigens, vSag7 and vSag9, which completely abrogated in vitro processing by convertases, were efficient in functional presentation to responsive T-cell hybridomas. This effect was observed in both endogenous presentation and paracrine transfer of the vSag. Processing by convertases thus appears not to be essential for vSag function. Finally, we have identified the purified endosomal protease cathepsin L as another protease that is able to cleave convertase mutant vSag in vitro, yielding fragments similar to those detected in vivo, thus suggesting that proteases other than convertases are involved in the activation of vSags.