The initial step in a humoral immune response involves the acquisition of antigens by B cells via surface immunoglobulin. Surprisingly, anatomic studies indicate that lymph-borne proteins do not have access to the follicles where naive B cells reside. Thus, it is unclear how B cells acquire antigens that drain to lymph nodes. By tracking a fluorescent antigen and a peptide:MHC II complex derived from it, we show that antigen-specific B cells residing in the follicles acquire antigen within minutes of injection, first in the region closest to the subcapsular sinus where lymph enters the lymph node. Antigen acquisition, presentation, and subsequent T cell-dependent activation did not require B cell migration through the T cell area or exposure to dendritic cells. These results indicate that the humoral response is initiated as soluble antigens diffuse directly from lymph in the subcapsular sinus to be acquired by antigen-specific B cells in the underlying follicles.