Fungicidal activity of Hst 5 is initiated by binding to cell surface proteins on Candida albicans, followed by intracellular transport to cytoplasmic effectors leading to cell death. As we identified heat shock 70 proteins (Ssa1p and/or Ssa2p) from C. albicans lysates that bind Hst 5, direct interactions between purified recombinant Ssa proteins and Hst 5 were tested by pull-down and yeast two-hybrid assays. Pulldown of both native complexes and those stabilized by cross-linking demonstrated higher affinity of Hst 5 for Ssa2p than for Ssa1p, in agreement with higher levels of interactions between Ssa2p and Hst 5 measured by yeast two-hybrid analyses. C. albicans ssa1Delta and ssa2Delta mutants were constructed to examine Hst 5 binding, translocation, and candidacidal activities. Both ssa1Delta and ssa2Delta mutants were indistinguishable from wild-type cells in growth and hyphal formation. However, C. albicans ssa2Delta mutants were highly resistant to the candidacidal activity of Hst 5, although the ssa1Delta mutant did not have any significant reduction in killing by Hst 5. Total cellular binding of 125I-Hst 5 in the ssa2Delta mutant was reduced to one-third that of wild-type cells, in contrast to the ssa1Delta mutant whose total cellular binding of Hst 5 was similar to the wild-type strain. Intracellular transport of Hst 5 was significantly impaired in the ssa2Delta mutant strain, but only mildly so in the ssa1Delta mutant. Thus, C. albicans Ssa2p facilitates fungicidal activity of Hst 5 in binding and intracellular translocation, whereas Ssa1p appears to have a lesser functional role in Hst 5 toxicity.