Spleen cells from adult specific pathogen-free miniature swine were exposed to sheep red blood cell (SRBC) 2 monolayers to follow the course of the immune response to immobilized particulate antigen. Spleen cells in medium were incubated for various time intervals on the monolayers, removed and cultured in new dishes without further exposure to SRBC antigen for 5.5 days. Spleen cells from unimmunized "normal" animals responded maximally with 6-h exposure to a monolayer consisting of 75% SRBC and 25% autologous pig red blood cells. Spleen cells from SRBC primed animals also responded optimally to monolayers of 75% SRBC but required only 2-h exposure before culturing. Only minimal numbers of SRBC were released from the monolayers, and carryover of antigen from the monolayer to the fresh tissue culture dishes was not a factor in promoting response. This method of in vitro immunization provides for pulse exposure to particulate antigen and facilitates precise evaluation of the role of antigen during the course of the immune response.