Adaptive B cell responses are initiated by B cell receptor-antigen engagement. Despite its importance, the early stages of B cell interaction with antigen in vivo remain elusive. Using fluorescent particulate antigen in combination with multiphoton microscopy and immune staining, we identified the follicle-subcapsular sinus (SCS) boundary of the lymph node as a site for B cell-antigen acquisition. In this macrophage-rich region, antigen accumulated shortly after administration, where specific follicular B cells were retained for long periods of time. These B cells acquired antigen cumulatively and became the main antigen carriers inside the follicle before polarizing to the B cell-T cell border. These observations not only provide evidence of a B cell role as antigen transporters into the follicle, but also highlight the boundary between the follicle and the SCS as a site for initiation of B cell responses.