Lymphocytes from guinea pigs having delayed hypersensitivity to horse-radish peroxidase (HRPO) when cultured in vitro with HRPO produce a large m.w. factor (greater than 100,000 daltons) that causes peritoneal macrophages from nonimmune animals to aggregate. The macrophage aggregation factor (MAF) can be separated from macrophage migration inhibitory factor (MIF) by gel filtration of active lymphocyte supernatants on Sephadex G-150. MAF is heat stable (56 degrees C for 30 min) but inactivated by trypsin. These data suggest that aggregation of macrophages in vitro by lymphokine-rich culture supernatants is not due to MIF but is caused by a separate large m.w. factor.