The haemagglutinin (HA) protein of fowl plague virus A/FPV/Rostock/34 (H7N1) contains three N-linked oligosaccharide side chains in its stem domain. These stem glycans, which are attached to the Asn residues at positions 12, 28 and 478, are highly conserved throughout all HA protein sequences analysed to date. In a previous study, in which mutant HA proteins lacking individual stem glycosylation sites had been expressed from an SV-40 vector, it was shown that these glycans maintain the HA protein in the metastable form required for fusion activity. In the present study, the functional role of the stem N-glycans for virus replication was investigated using recombinant influenza viruses generated by an RNA polymerase I-based system. Studies in Madin-Darby canine kidney cells and embryonated chickens' eggs revealed that the N-glycan at Asn(12) is crucial for virus replication. In both culture systems, growth of virus lacking this glycan (mutant cg1) was completely blocked at 37 degrees C and inhibited at 33 degrees C. Loss of the glycan from Asn(478) (mutant cg3) caused less striking, but still measurable, effects. Interestingly, it was not possible to generate mutant viruses containing the HA protein lacking the N-glycan at Asn(28). It is concluded from this that the N-glycan at Asn(28) is indispensable for the formation of replication-competent influenza viruses. When compared to viruses containing wild-type HA protein, mutants cg1 and cg3 showed a significantly decreased pH stability. Taken together, these data show that the HA stem glycans are potent regulators of influenza virus replication.