A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.