We have developed and characterized several murine cell lines that constitutively express either the full-length, membrane-bound form of influenza virus hemagglutinin (HA) or a truncated version of the protein (HAsec) that lacks the carboxyterminal anchoring sequences and is secreted from cells. cDNAs encoding HA or HAsec were linked to the murine metallothionein-I promoter or the SV40 early promoter, and inserted into plasmids containing the transforming DNA fragment of bovine papilloma virus (BPV). The resulting vectors were introduced into three cultured lines of murine cells--C127, NIH3T3 and MME--either alone or in the presence of a plasmid that carries the aminoglycoside transferase gene of Tn5. The resulting lines of MME cells contained 1-5 copies of the vector in an integrated state and expressed low levels of HA (approximately 10(4) molecules/cell). In contrast, lines of C127 and NIH3T3 cells were obtained that express up to 5 X 10(6) molecules of HA per cell or secrete approximately 10(7) molecules of HAsec per cell per 24 h. Some of these cell lines carry multiple (30-200) copies of the vector in an integrated state; in others, the vector is propagated as unit-length episomes or as oligomers. Both the membrane-bound and secreted forms of HA expressed in these cell lines display a more extensive pattern of glycosylation than HA or HAsec synthesized in simian cells and they are transported to the cell surface more slowly. Pulse-chase experiments suggest that the step which limits the rate at which HA and HAsec travel down the secretory pathway occurs in the rough endoplasmic reticulum before the molecules are transferred to the Golgi apparatus. Using indirect immunofluorescence in combination with a cell sorter, we have shown that the level of expression of HA within cloned populations of producing cells can be variable. However, greater than 90% of the cells in certain cell lines display considerable quantities of HA on their surface, as judged by their ability to bind red blood cells in large numbers. We have taken advantage of the membrane fusion activity of HA to effect the fusion of erythrocytes to these cells and to deliver the contents of red cell ghosts into the cells' cytoplasm.