To characterize the phenotypic and genotypic changes that occurred in a new clone lineage of Neisseria meningitidis (lineage III) in the Netherlands, the electrophoretic type (ET) was determined for 79 serogroup Bisolates of serotype 4 or subtype P1.4 (or both) obtained between 1958 and 1990 from patients with systemic meningococcal disease. Thirty-five previously described isolates were also included. After its appearance in 1980, lineage III started homogeneously with regard to both genotype (ET-24) and phenotype (B:4:P1.4). After 1984, other clones appeared in the lineage, and the various clones acquired other serotypes (serotypes 14 and 15) and subtypes (P1.2, P1.7, and P1.12), indicating frequent exchange of genetic material between clones. These results indicate that basing a serogroup B vaccine on outer membrane components from a single strain is not a valid strategy for the prevention of meningococcal disease.