The serum opacity factor (SOF) of Streptococcus pyogenes is a type-specific lipoproteinase of unknown biological significance. We have sequenced the sof gene and characterized the corresponding SOF protein from a strain of type M63. It was found that sof63 is related to sof22 and that, similar to SOF22 [25], SOF63 binds fibronectin. Moreover, we demonstrate opacity factor activity in a Streptococcus dysgalactiae fibronectin-binding protein FnBA that is structurally related to the SOF proteins of S. pyogenes. Sequence analysis of these three SOF proteins showed a unique periodical pattern of conserved and variable regions. The enzymatically active part of SOF63 was localized to the fragment corresponding to the entire set of conserved and variable sequences, while for fibronectin-binding a single repeat in the C terminal part of the protein was sufficient. The results show that streptococcal SOF proteins form a novel family of bifunctional proteins with lipoproteinase and fibronectin-binding activities.