The 28kDa outer membrane proteins (P28) of Ehrlichia chaffeensis are encoded by a multigene family. The purpose of this study was to determine all the p28 gene sequences and their transcriptional activities. There were 21 members of the p28 multigene family located in a 23kb DNA fragment in the genome of E. chaffeensis. The p28 genes each contained 816-903 nucleotides with intergenic spaces of 10-605 nucleotides. All the genes were complete and were predicted to have a signal sequence. The molecular masses of the mature proteins were predicted to be 28-32kDa. The amino acid sequence identity of the P28 proteins was 20-83%. Ten p28 genes were investigated for transcriptional activity by using RT-PCR amplification of mRNA. Six of 10 tested p28 genes were actively transcribed in cell-culture grown E. chaffeensis. RT-PCR also indicated that each of the p28 genes was monocistronic. These results suggest that the p28 genes are active genes and encode polymorphic forms of the P28 proteins. The P28s were divergent among isolates of E. chaffeensis also. The large repertoire of the p28 genes in a single ehrlichial organism and antigenic diversity of the P28 among the isolates of E. chaffeensis suggest that P28s may be involved in immune avoidance.