Nucleotide sequences of the neuraminidase (NA) genes of 33 influenza A (H3N2) epidemic strains isolated between 1968 and 1995 were analyzed to determine their evolutionary relationships. Phylogenetic analysis using the DNA maximum-likelihood method indicates that the NA genes of recent H3N2 field strains, like their hemagglutinin genes (HA), have evolved as two distinct lineages represented by the vaccine strains. A/Beijing/353/89 and A/Beijing/32/92 for A/Shanghai/24/ 90). Furthermore, genetic reassortment of NA genes between the two lineages occurred during their circulation. Genetic reassortants, which bear an A/Beijing/32/92-like HA and an A/Beijing/353/89-like NA, have circulated worldwide and are representative of current influenza A (H3N2) epidemic strains. The mutation rate of the NA gene was found to be 2.28 x 10(-3) per nucleotide site per year with 4.2% of the mutations resulting in amino acid substitutions. Thirty-five percent of the amino acid substitutions was located in sites previously suggested to be reactive to antibody. Amino acid residues involved in NA enzyme activity have been conserved. Seven potential glycosylation sites identified in the NA of A/Hong Kong/8/68 virus were conserved by the majority of isolates, with more recently circulating viruses having an additional glycosylation site. Comparison of the rate of amino acid substitutions in the NA stalk to that of entire NA revealed high variability in this region. These findings demonstrate the importance of closely monitoring both the HA and the NA genes of influenza viruses to aid vaccine strain selection.