The synthesis of Semliki Forest virus nucleocapsid in infected cells was studied by labelling the virus RNAs with 3H-uridine for different periods at various phases of infection. Short pulses (10 to 20 min) revealed the accumulation of 42S RNA in a ribonucleoprotein which sedimented at about 90S (90S RNP) and contained only small amounts of capsid protein. Only after longer pulses was the labelled 42S RNA found in the virus nucleocapsid, suggesting that the 90S RNP may be its precursor. The life time of the 90S RNP was long in the early phases of infection and short in the late phases, reflecting the increased rate of assembly of the nucleocapsid during infection. The 90S RNP was the only 42S RNA containing RNP found in cells infected with temperature sensitive mutants deficient in nucleocapsid formation or wild type infected cells treated with cycloheximide to inhibit nucleocapsid assembly.