A serum-free medium formulation that allows a primary in vitro antibody response to the nonmitogenic thymic independent antigen TNP-AECM-Ficoll is described. Purified murine B lymphocytes and adherent accessory cells are required for optimal specific responses as well as for optimal B cell survival. The medium is based on Iscove's modification of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture. The serum-replacing components are insulin, transferrin, and progesterone. The survival of unstimulated murine T cells in this Iscove/F12 medium is poor, but B cells survive for relatively long periods of time. This medium should prove useful in defining the molecular requirements for B lymphocyte activation by antigen.