Highly pure populations of human T and B lymphocytes were prepared from tonsils using a two‐step fractionation procedure. Cells were first fractionated on discontinuous gradients of bovine serum albumin, then separated on the basis of their reactivity with the sheep red cell intermediates, E and EACl423. More than 95 % of the cells in the final suspensions were either T or B lymphocytes. Phytohemagglutinin and concanavalin A were mitogenic exclusively for T cells. Pokeweed was mitogenic for both T and B cells. T cell proliferation occurred in response to two antigens to which the cell donors were immune: tetanus toxoid (TT) and polyribosephosphate (PRP) capsular antigen of Hemophilus influenzae, type b. B lymphocytes did not transform in response to either PRP, soluble TT, or insoluble TT, but transformed vigorously in the presence of supernatants obtained from TT‐activated T cells.