Cloned cDNA encoding the membrane glycoprotein E1 of the coronavirus mouse hepatitis virus strain A59 was expressed transiently in a monkey fibroblast cell line (COS) by using a simian virus 40-based vector. As determined by indirect immunofluorescence microscopy, the E1 protein accumulated intracellularly in a perinuclear region coincident with a Golgi marker. The same three species of E1 that occur in virus-infected cells were also found in transfected cells. These are one unglycosylated form and two apparently O-glycosylated forms that could be labeled in a tunicamycin-resistant fashion with [3H]glucosamine. Because O glycosylation occurs posttranslationally in the Golgi apparatus, we could show, by monitoring the rate of acquisition of oligosaccharides, that the transport of E1 from the rough endoplasmic reticulum to the Golgi apparatus had a half time of between 15 and 30 min.