Summary A host-mediated assay system using mouse leukemic L 5178Y cells was used for studies of known mutagenic agents. This forward mutagenesis system utilizes resistance to methotrexate (MTX), cytosine arabinoside (ara-C) and high concentrations of thymidine (TdR) to serve as genetic markers. Sub-lines of L 5178Y cells specially selected for mutagenesis studies possess a high infectivity in genetically compatible mice and grow in vivo exponentially up t0 approx. 10 8 cells/ml in ascitic fluids with a doubling time of 14 h. The cells also grow in vitro exponentially with high cloning efficiencies (40-90%). The effectiveness 0f the system in detecting mutagens was tested with known mutagens (ethyl methanesulfonate (EMS) and 5-bromodeoxyuridine (BUdR)). Both compounds increased mutant frequencies as the result of the induction of mutation in the host-mediated assay as well as in vitro. Dose-response experiments showed that the increase in mutant frequency was a function of the levels of the mutagen employed. Mutant induction in the host was obtained without loss of viability of target cells or weight loss of the animals.