Surface proteins of Gram-positive bacteria are covalently linked to the cell wall envelope by a mechanism requiring an N-terminal signal peptide and a C-terminal LPXTG motif sorting signal. We show here that surface proteins of Staphylococcus aureus arrive at two distinct destinations in the bacterial envelope, either distributed as a ring surrounding each cell or as discrete assembly sites. Proteins with ring-like distribution (clumping factor A (ClfA), Spa, fibronectin-binding protein B (FnbpB), serine-aspartate repeat protein C (SdrC) and SdrD) harbour signal peptides with a YSIRK/GS motif, whereas proteins directed to discrete assembly sites (S. aureus surface protein A (SasA), SasD, SasF and SasK) do not. Reciprocal exchange of signal peptides between surface proteins with (ClfA) or without the YSIRK/GS motif (SasF) directed recombinant products to the alternate destination, whereas mutations that altered only the YSIRK sequence had no effect. Our observations suggest that S. aureus distinguishes between signal peptides to address proteins to either the cell pole (signal peptides without YSIRK/GS) or the cross wall, the peptidoglycan layer that forms during cell division to separate new daughter cells (signal peptides with YISRK/GS motif).