Nonsynchronized and hydroxyurea (HU)-synchronized SV40-transformed human cells (W98VaD) were fused with chick embryo erythrocytes (CE). The uptake of T antigen by CE nuclei was compared with initiation of chick nuclear DNA synthesis. Uptake of T antigen by CE nuclei occurred at about the same time after fusion with asynchronous as with HU-synchronized cells. CE nuclei rapidly became T antigen-positive between 16 h and 28 h after fusion and usually almost all CE nuclei were T antigen-positive by 48 h after fusion. In contrast, initiation of chick nuclear DNA synthesis occurred as a function of time after reversal of the HU block, when the host cell nuclei were also synthesizing DNA. Chick nuclear DNA synthesis occurred in many heterokaryons before the CE nuclei became T antigen-positive by immunofluorescence.