Proliferation was observed during in vitro cultivation of peritoneal exudate cells that had been educed from a C3H mouse with Freund's incomplete adjuvant. These cells were successfully subcultured by release with trypsin-EDTA solution and are now at passage 108 after 22 months in culture. Using this technique, 12 other rapidly growing peritoneal exudate cultures were obtained, whereas 10 cultures not educed with adjuvant did not proliferate. Characteristics of four adjuvant-induced cell lines established in culture include: rapid attachment to glass, doubling time in culture of 18 to 19 hr, phagocytosis of colloidal carbon, enhanced phagocytosis of specifically sensitized bacteria, epithelium-like morphology, and retention of C3H histocompatible specificities. These cell lines had widely varying chromosome distributions with modes from 37.3 +/- 2.4 to 82.6 +/- 2.30, but inoculation of 10(7) cultured cells into syngeneic animals did not produce tumors. Procedures described for the reproducible establishment of peritoneal exudate cell lines did not require use of conditioned media or exogenous viral infection.