To identify rate-limiting steps in T cell-independent type 2 antibody production against polysaccharide antigens, we performed a genome-wide screen by immunizing several hundred pedigrees of C57BL/6 mice segregating N-ethyl-N-nitrosurea-induced mis-sense mutations. Two independent mutations, Tilcara and Untied, were isolated that semi-dominantly diminished antibody against polysaccharide but not protein antigens. Both mutations resulted from single-amino-acid substitutions within the kinase domain of protein kinase C-β (PKCβ). In Tilcara, a Ser552>Pro mutation occurred in helix G, in close proximity to a docking site for the inhibitory N-terminal pseudosubstrate domain of the enzyme, resulting in almost complete loss of active, autophosphorylated PKCβI, whereas the amount of alternatively spliced PKCβII protein was not markedly reduced. Circulating B cell subsets were normal and acute responses to B-cell receptor stimulation such as CD25 induction and initiation of DNA synthesis were only measurably diminished in Tilcara homozygotes, whereas the fraction of cells that had divided multiple times was decreased to an intermediate degree in heterozygotes. These results, coupled with evidence of numerous mis-sense PRKCB mutations in the human genome, identify Prkcb as a genetically sensitive step likely to contribute substantially to population variability in anti-polysaccharide antibody levels.