The feasibility of a linear expression cassette (LEC)-based influenza A DNA vaccine was demonstrated in mice, using a lethal dose (LD90) of a mouse-adapted A/Hong Kong/8/68 (H3N2) influenza strain. LECs expressing hemagglutinin (HA) from either the homotypic H3N2 or the heterotypic H1N1 (A/Puerto Rico/8/34) influenza virus were produced by polymerase chain reaction and either phosphodiester- or phosphorothioate-modified oligonucleotide primers. Survival subsequent to lethal viral challenge was used as a primary end point; weight loss was the secondary end point. Survival and weight loss data showed that protection can be achieved in mice with 50 microg of phosphate-buffered saline-formulated LEC DNA or 2 microg of Vaxfectin-formulated LEC DNA. Survival correlated with neutralizing antibody titers (hemagglutination inhibition, HAI); titers obtained after vaccination with LEC were equivalent to those obtained with HA (H3N2) plasmid DNA control. Vaccination with heterotypic H1 HA-LEC DNA provided no protection against viral challenge.