We have analyzed the intracellular transport of the spike (S) protein of infectious bronchitis virus (IBV), an avian coronavirus. Surface expression was analyzed by immunofluorescence microscopy, by surface biotinylation, and by syncytium formation by S-expressing cells. By applying these methods, the S protein was found to be retained intracellularly. Tyr1143 in the cytoplasmic tail was shown to be a crucial component of the retention signal. Deletion of a dilysine motif that has previously been suggested to function as a retrieval signal did not abolish intracellular retention. Treatment of the S proteins with endoglycosidases did not reveal any differences between the parental and the mutant proteins. Furthermore, all S proteins analyzed were posttranslationally cleaved into the subunits S1 and S2. In coexpression experiments, the S protein was found to colocalize with a Golgi marker. Taken together, these results indicate that the S protein of IBV is retained at a late Golgi compartment. Therefore, this viral surface protein differs from the S proteins of transmissible gastroenteritis virus and severe acute respiratory syndrome coronavirus, which are retained at a pre-Golgi compartment or transported to the cell surface, respectively. The implications of these differences are discussed.