Sortases are cysteine transpeptidases that are essential for the assembly and anchoring of cell-surface adhesins in Gram-positive bacteria. In Streptococcus agalactiae (GBS), the pilin-specific sortase SrtC1 catalyzes the polymerization of pilins encoded by pilus island 1 (PI-1) and the housekeeping sortase SrtA is necessary for cell-wall anchoring of the resulting pilus polymers. These sortases are known to utilize different substrates for pilus polymerization and cell-wall anchoring; however, the structural correlates that dictate their substrate specificity have not yet been clearly defined. This report presents the expression, purification and crystallization of SrtC1 (SAG0647) and SrtA (SAG0961) from S. agalactiae strain 2603V/R. The GBS SrtC1 has been crystallized in three crystal forms and the GBS SrtA has been crystallized in one crystal form. © 2010 International Union of Crystallography All rights reserved.