SUMMARY The replication of rabbitpox virus has been studied in mouse sarcoma 18o cells in media containing either arginine or citrulline. The yield of infective virus depen- ded on the concentration of each amino acid, and maximum yield was obtained with o.I mM-arginine or o'5 mM-citrulline. In the presence of this concentration of citrulline, cell multiplication and protein synthesis in uninfected cells was suppressed markedly compared with cell cultures in medium containing 0"5 mM-arginine. Growth of a human citrullinaemia cell line was inhibited also when citrulline substituted for arginine yet the replication of vaccinia virus continued. The incorporation of radioactivity available as (l~C)-carbamoyl-citrulline into mouse sarcoma I8o cells maintained in citrulline-containing medium increased significantly following infection by rabbitpox virus. A similar, increased incorpora- tion was observed with vaccinia virus-infected HeLa cells. Increased incorporation into rabbitpox virus-infected mouse sarcoma I8O ceils occurred at 3 and 5 h post- infection and coincided with increased incorporation of (14C)-guanido-arginine into cell cultures infected in the presence of arginine. This enhanced utilization of citrulline was inhibited by actinomycin D, and rabbitpox virus replication in the presence of citrulline was inhibited by canavanine. It is concluded that virus- directed mechanisms determine arginine biosynthesis in mouse sarcoma I8o cells infected with rabbitpox virus in the presence of citrulline.