A previously identified nucleoside triphosphatase activity in mammalian reovirus cores was further characterized by comparing two reovirus strains whose cores differ in their efficiencies of ATP hydrolysis. In assays using a panel of reassortant viruses derived from these strains, the difference in ATPase activity at standard conditions was genetically associated with viral genome segment L3, encoding protein lambda1, a major constituent of the core shell that possesses sequence motifs characteristic of other ATPases. The ATPase activity of cores was affected by several other reaction components, including temperature, pH, nature and concentration of monovalent and divalent cations, and nature and concentration of anions. A strain difference in the response of core ATPase activity to monovalent acetate salts was also mapped to L3/lambda1 by using reassortant viruses. Experiments with different nucleoside triphosphates demonstrated that ATP is the preferred ribonucleotide substrate for cores of both strains. Other experiments suggested that the ATPase is latent in reovirus virions and infectious subviral particles but undergoes activation during production of cores in close association with the protease-mediated degradation of outer-capsid protein mu1 and its cleavage products, suggesting that mu1 may play a role in regulating the ATPase.