We have studied the conditions required for the activation in vitro of memory B cells generated in vivo. BALB/c mice were immunised by a single injection of antigen emulsified in Freund's complete adjuvant. Splenocytes were isolated after different time intervals and cultured in a serum-free medium in the presence of antigen and thymocyte-conditioned medium. After 3 days the splenocytes were fused with myeloma cells. A minimum time interval of more than 2 weeks between priming in vivo and stimulation in vitro was required in order to obtain antigen-specific IgG-secreting hybridomas. After a time interval of 4 weeks or longer most of the antigen-specific hybridomas secreted IgGs. During stimulation in vitro the presence of antigen and of T cells was found to be essential for obtaining an antigen-specific IgG response. The addition of thymocyte-conditioned medium enhanced the IgG response.