Vaccination with recombinant (r) hsp60 protects mice against Histoplasma capsulatum. To map the domain of rhsp60 that confers protection, four gene segments were cloned into pET19b and expressed in Escherichia coli. rhsp60 fragments were tested for their capacity to induce proliferation by sensitized splenocytes and to protect BALB/c and C57BL/6 mice. All polypeptides stimulated cells from BALB/c mice immunized with yeasts or with rhsp60. Fragment 3 caused the most vigorous response by cells from mice immunized yeasts, and fragment 1 caused the most vigorous response by cells from mice immunized with rhsp60. Splenocytes from yeast-immunized C57BL/6 mice did not recognize any polypeptide. In contrast, cells from C57BL/6 mice inoculated with rhsp60 responded to all fragments but most intensely to fragment 2. In both strains, fragment 3 conferred protection against sublethal and lethal challenges. Thus, a common protective domain of hsp60 has been identified.