The GapC products of Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis share considerable homology at the DNA and amino acid levels. The high similarity at the protein level suggests that one GapC protein might be used as a single antigen to protect dairy cows against infections with the contagious S. agalactiae and the environmental S. dysgalactiae and S. uberis strains. Despite their similarities, immunization with the S. dysgalactiae GapC did not protect dairy cows from a challenge with S. uberis, suggesting the presence of regions in GapC that are involved in species-specific protection. To produce a single antigen that can be used to protect against all streptococcal mastitis infections, we constructed a GapC chimeric protein using the S. uberis GapC product as the backbone followed by non-conserved peptide regions from the S. agalactiae and S. dysgalactiae GapC proteins. We report that the chimeric GapC protein retains the enzymatic activity of the S. uberis GapC protein. In addition, we fused the chimera to the OmpF and LipoF transport sequences of Escherichia coli and the GapC chimeras were present in membrane fractions of E. coli. These extracts could be the basis of an antigen preparation for use in mastitis vaccines.