The amino acid sequences of eight variants of thermostable direct hemolysin (TDH) and a TDH-related hemolysin, deduced from nucleotide sequences, were compared. Fourteen amino acid residues, mainly within conserved regions, were chosen to prepare mutant TDHs with one or two amino-acid replacements by site-directed mutagenesis. Of the 25 mutant TDHs prepared, those with replacements of Trp65 (Trp65-His65, Trp65-Tyr65, Trp65-Leu65) or Leu66 (Leu66-Ser66) showed very significant reduction (more than 150-fold) of hemolytic activity. Several other mutant TDHs with replacements at Lys45, Arg46 and Gly90 showed weaker hemolytic activity than the wild-type TDH, but extents of reduction in hemolytic activity were much less than for mutant TDHs with replacements at Trp65 and Leu66. These results indicate that Trp65 and Leu66 are very important for the hemolytic activity of TDH.