The polar pili of Pseudomonas aeruginosa consist of a subunit protein, pilin, which is a 144-residue polypeptide that contains a hydrophobic N-terminal region and eight hydrophilic regions distributed throughout the remainder of the molecule. T cells from mice immunized with pili or whole bacteria gave good pilus-specific T-cell proliferation responses. To delineate the T-cell antigenic regions of the pilin, T-cell blasts were generated from lymph nodes of pilus-primed BALB/c mice. These blasts were tested in vitro in T-cell proliferation assays for reactivity against the fragments of the pilin subunit prepared by enzymatic digestion. Citraconylation followed by trypsin digestion (cT) of the pilin subunit cleaved the protein into four fragments, cTI (residues 1 to 30), cTII (residues 31 to 53), cTIII (residues 54 to 120), and cTIV (residues 121 to 144). The ability to stimulate the T cells was found to reside in the cTI and cTIII regions, but not in the cTII or cTIV regions. A subfragment of cTIII, containing residues 82 to 104, was identified as the major T-cell recognition site within the cTIII region of the pilin molecule. A cross-reactivity was observed between pili from two strains of P. aeruginosa, namely, PAK and PAO, at the T-cell level. This cross-reactivity probably resulted from the sequence homology in the hydrophobic N-terminal region of these two molecules.