Summary We have studied several aspects of the inhibition of DNA synthesis in Mengovirus-infected cells. In mouse L-929 cells at 5 h after infection at a multiplicity of 200 p.f.u./cell, there was a decrease in DNA synthesis. However, the rate of DNA replication fork movement, measured by equilibrium sedimentation of DNA sequentially labelled with BrdUrd and 3H-thymidine, was not significantly reduced in infected cells despite an 84% inhibition of DNA synthesis. Enzymic assay of the total cellular dTTP pool size showed it to be increased 37% in infected cells compared to controls. This was not accounted for by enhanced entry of thymidine nucleotides into the pool from either exogenous or endogenous sources, since the buoyant density of DNA pulse-labelled with 3H-BrdUrd was the same in infected and uninfected cells and there was no evidence for breakdown of DNA in infected cells. There was also a moderate decrease in the uptake of exogenous thymidine into the cell, shown by a reduction in the Vmax for transport with little change in the K m. By 6 h after infection, the dTTP pool size was slightly smaller in infected cells than in the controls and there was a marked inhibition in the rate of replication fork movement. These results show that Mengovirus infection blocks the entry of dTTP into DNA. At 5 h after infection, this block is manifested by an inhibition of initiation of new DNA chain synthesis. By 6 h, replication fork movement is also retarded and there is a more generalized derangement in DNA synthesis.