The activity of histatin 5 (Hst 5) against Candida albicans is initiated through cell wall binding, followed by translocation and intracellular targeting. The C. albicans cell wall protein Ssa2 is involved in the transport of Hst 5 into cells as part of cell killing. P-113 (a 12-amino-acid candidacidal active fragment of Hst 5) and P-113Q2.10 (which is inactivated by a glutamine substitution of the Lys residues at positions 2 and 10) were compared for their levels of cell wall binding and intracellular translocation in Candida wild-type (wt) and ssa2Delta strains. Both P-113 and P-113Q2.10 bound to the walls of C. albicans wt and ssa2Delta cells, although the quantity of P-113Q2.10 in cell wall extracts was higher than that of P-113 in both strains. Increasing the extracellular NaCl concentration to 100 mM completely inhibited the cell wall association of both peptides, suggesting that these interactions are primarily ionic. The accumulation of P-113 in the cytosol of wt cells reached maximal levels within 15 min (0.26 microg/10(7) cells), while ssa2Delta mutant cells had maximal cytosolic levels of less than 0.2 microg/10(7) cells even after 30 min of incubation. Furthermore, P-113 but not P-113Q2.10 showed specific binding with a peptide array of C. albicans Ssa2p. P-113Q2.10 was not transported into the cytosol of either C. albicans wt or ssa2Delta cells, despite the high levels of cell wall binding, showing that the two cationic lysine residues at positions 2 and 10 in the P-113 peptide are important for transport into the cytosol and that binding and transport are independent functional events.