Gs protein is a shorter, soluble form of the viral G protein of vesicular stomatitis virus (VSV) lacking the membrane-anchoring domain. Production of Gs protein appears to be a general property of VSV because infection of BHK-21 cells by five different isolates of the VSV serotype Indiana led in all cases to the synthesis of Gs protein. Moreover, it is formed in a variety of eucaryotic cell lines after VSV infection. In pulse-chase experiments, we observed a time-dependent change in the ratio of G to Gs protein released into the growth medium, suggesting that Gs is formed intracellularly rather than on the cell surface. Further experiments revealed that Gs protein can be synthesized in vitro in the reticulocyte lysate system after addition of a viral mRNA fraction and in a coupled transcription-translation system with VSV core particles. In the presence of microsomal membranes both G and Gs protein were glycosylated in the reticulocyte lysate, confirming that the authentic Gs protein is synthesized in vitro. The addition of various protease inhibitors to the cell-free system and variation of the incubation conditions did not alter the ratio of G to Gs formation. Taken together, these experiments suggest strongly that Gs protein is not a product of a membrane-associated proteolytic activity but is formed during or shortly after the translation process. Our attempts to detect a specific, shorter mRNA coding for the Gs protein by molecular hybridization procedures did not reveal the existence of such a mRNA species.