The kinetics of cleavage of pr92gp, the precursor of the two glycoproteins of Rous sarcoma virus gp85 and gp35, were followed. Viral glycoproteins were detected by immunoprecipitation with anti-gp85 and anti-gp35 serum. It could be shown in pulse-chase experiments that little or no intracellular cleavage of the precursor took place during the time in which the majority of newly synthesized viral glycoprotein was exported from the cells. Soon after its synthesis, however, pr92gp underwent some modification that made it migrate slightly faster on sodium dodecyl sulfate-polyacrylamide gels. Under steady state conditions the precursor was shown to be the predominant form of intracellular viral glycoprotein. Virus which was harvested every 2 min from infected cells prelabeled for 90 min with [3H]mannose contained mostly uncleaved and only a little mature glycoprotein. By incubation of this freshly released virus in serum-free buffer, the majority of the glycoprotein precursor could be cleaved into mature gp85 and gp35. Virus harvested every 10 min contained only mature glycoproteins.