Salivary histatins are a family of small histidine-rich peptides with potent antifungal activity. We previously identified a 70-kDa cell envelope protein in Candida albicans and Saccharomyces cerevisiae that mediates binding of histatin (Hst) 5. Isolation of Hst 5-binding protein followed by matrix-assisted laser desorption ionization mass spectrometry analysis identified this protein as the heat shock protein Ssa1p. Ssa protein and Hst 5-binding protein were found to be co-localized on immunoblots of yeast beta-mercaptoethanol cell wall extracts and cytosolic fractions. Yeast two-hybrid analysis showed strong interactions between Ssa1p and both Hst 3 and Hst 5. To assess functional roles of Ssa proteins in the Hst 5 antifungal mechanism in vivo, both binding and fungicidal assays were carried out using S. cerevisiae isogenic SSA1/SSA2 mutants. 125I-Hst 5 binding assays showed saturable binding (Kd = 2.57 x 10(-6) m) with the wild-type SSA1/SSA2 strain; however, Hst 5 binding with the Deltassa1ssa2 double mutant was reduced (Kd = 1.25 x 10(-6) m). Cell wall HSP70 proteins were also diminished, but still detectable, in S. cerevisiae Deltassa1ssa2 cells and are likely to be Ssa3p or Ssa4p. Hst 5 (31 microm) killed 80% of the wild-type cells in fungicidal assays at room temperature. However, only 50-60% killing of the single mutants (Deltassa1 and Deltassa2) was observed, and fungicidal activity was further reduced to 20-30% in the Deltassa1ssa2 double mutant. Incubation of cells under heat shock conditions increased the sensitivity of cells to Hst 5, which correlated with increased Hst 5-binding activity in Deltassa1ssa2 cells, but not in wild-type cells. This study provides evidence for a novel function for yeast Ssa1/2 proteins as cell envelope binding receptors for Hst 5 that mediate fungicidal activity.