A fraction of thymic lymphomas induced by high LET neutron radiation contains activating mutations (single-base substitutions) in the ras genes. To determine whether such mutations are the result of the interaction of high LET radiation with cellular DNA, we have utilized an in vitro model system to screen and isolate neutron-radiation-induced mutants. With that aim, we irradiated the PL6 I hamster cell line with 0.4 MeV neutrons. This cell line contains linked copies of the gpt and neor genes, which permits selection for large or small alterations, depending on the selection imposed. Mutants selected for large alterations represented 98.2% of the total. When selection for small mutations was imposed, 9 clones grew. The molecular and biochemical analysis of these clones revealed that 5 of them had identifiable mutations in the gpt gene, consisting of small insertions and deletions, but no single-base substitutions were detected. This represents the first sequence characterization of neutron-induced mutants. The results obtained are consistent with the notion that the ras point mutations identified in the neutron-induced tumors are most likely detected due to the strong selective advantage that they confer to the host cell, but they probably arose during tumor evolution, since they represent a negligible proportion of the total number of alterations induced by neutron radiation.