Opacity (Opa) proteins are a family of antigenically variable outer-membrane proteins of Neisseria meningitidis. Even among clonally related epidemic meningococcal isolates, there is greater variation of Opa protein expression than can be accounted for by the opa gene repertoire of any individual strain. We characterized the opa genes of eight closely related isolates of serogroup A N. meningitidis (subgroup IV-1) from a recent meningitis epidemic in West Africa. DNA sequence analysis and Southern blot experiments indicated that changes occurred in the opa genes of these bacteria as they spread through the human population, over a relatively short period of time. Such changes in one or a few loci within a clonal population are referred to as microevolution. The distribution of sequences present in hypervariable (HV) regions of the opa genes suggests that duplication of all or part of opa genes into other opa loci changed the repertoire of Opa proteins that could be expressed. Additional variability in this gene family appears to have been introduced by horizontal exchange of opa sequences from other meningococcal strains and from Neisseria gonorrhoeae. These results indicate that processes of recombination and genetic exchange contributed to variability in major surface antigens of this clonal population of pathogenic bacteria.