The incubation fluid from 24-hr cultures of normal guinea pig small intestines was found to contain activity capable of inhibiting the migration of normal guinea pig peritoneal macrophages. Each of three different culture fluids showed macrophage inhibitory activity in the 25,000-55,000 mol wt range when subjected to Sephadex G-100 gel filtration. One of these three preparations also contained inhibitory activity in the excluded volume. The low molecular weight activity was nondialyzable and heat stable to 56 degrees for 30 min. It was destroyed by boiling for 1 hr, and its activity was reversed by 10(-4)M epinephrine. Based upon these properties it is suggested that this activity was due to migration inhibitory factor (MIF). The high molecular weight inhibitor was stable to heating and its activity was not reversed by epinephrine. This material was most likely endotoxin. The presence of MIF, which may be associated with T-cell activity in the intestine, suggests that cell-mediated immunity (CMI) could play a role in local protection against gut infections. Furthermore, its presence in normal intestinal secretions suggests that some MIF-producing cells are always being stimulated by normal flora. The question is thus raised whether further specific stimulation of local CMI in the gut could be successful.