Peritoneal fluid from the abdominal cavities of guinea pigs having delayed hypersensitivity to horseradish peroxidase (HRPO) was obtained by a lavage technique before and after i.p. challenge with antigen. Macrophage migration inhibitory factor (MIF) and macrophage chemotactic factor activities were measured in peritoneal fluids from each animal. Chemotactic activity for macrophages was maximal 24 hr after i.p. challenge and was absent thereafter. MIF activity was maximal in peritoneal fluid 24 to 48 hr after challenge. Macrophages were present in greatest numbers in peritoneal fluid 24 hr after challenge and returned almost to control levels at 48 hr. Macrophages in 48-hr fluid were larger and exhibited more intense cytoplasmic staining for nonspecific esterases when compared to those in 0-hr fluid. The m.w. of MIF obtained from culture supernatants of HRPO-stimulated guinea pig lymphocytes and 48-hr peritoneal fluid were found to be virtually identical, 58,000 and 54,000 daltons, respectively. MIF from these in vitro and in vivo sources were similarly resistant to heating at 56 degrees C for 30 min but were both destroyed by incubation with isoluble trypsin.