Spleen cells from germfree rats, conventionally reared rats, and gnotobiotic rats associated with two Pseudomonas species gave no positive blastogenic response when incubated with each of four lipopolysaccharide (LPS) preparations from Escherichia coli, with glycolipid extracted from Salmonella minnesota R595 or with S. minnesota R595 lipid A. However, spleen cell preparations from athymic mice demonstrated a positive blastogenic response when incubated with E. coli LPS. Removal of adherent cells from germfree and conventional-flora rat spleen cells did not increase the mitogenic activity of LPS for nonadherent cells (less than 0.5% esterase-positive cells). All rat spleen cell preparations gave positive blastogenic responses to phytohemagglutinin and concanavalin A. This study indicates that LPS may not be a mitogenic agent for rat spleen cells.