We previously reported that, in human heat shock protein (Hsp) 90 (hHsp90), there are 4 highly immunogenic sites, designated sites Ia, Ib, Ic, and II. This study was performed to further characterize their epitopes and to identify the epitope that is potentially common to all members of the Hsp90 family. Panning of a bacterial library carrying randomized dodecapeptides revealed that Glu₂₅₁-Ser-X-Asp₂₅₄ constituted site Ia and Pro₂₉₅-Ile-Trp-Thr-Arg₂₉₉, site Ic. Site II (Asp₇₀₁-Pro₇₁₇) was composed of several epitopes. When 19 anti-hHsp90 monoclonal antibodies (mAbs) were subjected to immunoblotting against recombinant forms of 7 Hsp90-family members, 2 mAbs (K41110 and K41116C) that recognized site Ic bound to yeast Hsp90 with affinity identical to that for hHsp90, and 1 mAb (K3729) that recognized Glu₂₂₂-Ala₂₃₁ of hHsp90β could bind to human 94-kDa glucose-regulated protein (Grp94), an endoplasmic reticulum paralog of Hsp90. Among the 5 amino acids constituting site Ic, Trp₂₉₇ and Pro₂₉₅ were essential for recognition by all anti–site-Ic mAbs, and Arg₂₉₉ was important for most of them. The necessity of Ile₂₉₆, Thr₂₉₈, and Arg₂₉₉, which are replaced by Leu, Met/Leu, and Lys, respectively, in some eukaryotic Hsp90, was dependent on the mAbs, and K41110 and K41116C could react with Hsp90s carrying these substitutions. From these data taken together, we propose that the pentapeptide Pro₂₉₅-Ile-Trp-Thr-Arg₂₉₉ of hHsp90 functions as an immunodominant epitope common to all eukaryotic Hsp90.