Streptococcus agalactiae (group B streptococci [GBS]) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C proteins of GBS play a role in immunity, but their number, size, structure, function, and virulence properties have not been well characterized. A recombinant library of DNA fragments from GBS strain A909 (type Ia/C) was prepared in the plasmid pUX12, a specially constructed Escherichia coli expression vector. The library was screened with a rabbit antiserum shown to be protective for passive immunity to GBS infection in a mouse lethality model. Clones were divided into two distinct groups on the basis of DNA-DNA cross-hybridization, restriction enzyme analysis, and the expression of antigenic proteins in E. coli. A characteristic clone from each group was chosen for further study. Clone pJMS23 expresses gene products that biochemically and immunologically correspond to the trypsin-resistant, C-protein alpha antigen. Clone pJMS1 expresses a gene product that binds to immunoglobulin A and is similar to the trypsin-sensitive, C-protein beta antigen. Antisera raised in rabbits against E. coli containing each of the plasmid clones were able to elicit protective immunity in mice challenged by GBS strains carrying the C proteins but not by non-C-protein-bearing strains. Southern blot analysis shows no DNA homology between the clones, and there is no immunological cross-reactivity between the antigens they express. Therefore, pJMS23 and pJMS1 encode two different C proteins that define unique protective epitopes.