The transport kinetics of the influenza virus hemagglutinin from its site of synthesis to the apical plasma membrane of Madin-Darby canine kidney cells, a polarized epithelial cell line, were studied by a sensitive tryptic assay. Hemagglutinin acquired terminal sugars, as judged by sensitivity to endo-@-N-acetylglucosaminidase H, lo-15 min after synthesis, and first appeared on the apical domain 15 min later. None of the pulse-labeled hemagglutinin accumulated on the basolateral domain. At 20 C, terminal glycosylation continued, but no hemagglutinin was detected on the cell surface within 2 hr. If the incubation temperature was raised from 20 C to 37OC, hemagglutinin was quickly externalized, demonstrating that the inhibition at low temperature was reversible.