The influenza A virus hemagglutinin (HA) has three conserved oligosaccharides located in the stem region at asparagine residues 12, 28, and 478. The biological role of these oligosaccharides has been investigated by mutational analysis of HA of fowl plague virus that was expressed from a simian virus 40 vector in the presence of ammonium chloride for protection from acid denaturation in the trans-Golgi network. Resistance to endoglycosidase H and cleavage of HA into the subunits HA1 and HA2 have been analyzed as markers for intracellular transport. Cell surface exposure has been determined by hemadsorption following neuraminidase treatment, by immunofluorescence staining, and by fluorescence-activated cell sorter analysis. When all three stem oligosaccharides were removed, transport was almost completely blocked. When two of the three stem oligosaccharides, particularly those at asparagine residues 12 and 28, were missing, HA was transported to the surface but showed extremely low fusion activity. With mutants lacking one stem oligosaccharide, fusion was reduced to a lesser extent. Removal of stem oligosaccharides resulted also in an increase in the pH optimum required for fusion. On the other hand, no reduction in fusion activity was observed when oligosaccharides in the head region of the HA spike were removed. These results indicate that the conserved oligosaccharides in the stem stabilize HA in the form susceptible to the conformational change necessary for fusion.