Sequence　alignment　has　been　a　most　commonly　used　method　in　modern　biological　research.　However,　Sequence　alignment　in　＇Twilight　Zone＇　is　still　a　challenging　issue.　Three　protein　class-specific　matrices　are　constructed　to　investigate　whether　alignment　in　＇Twilight　Zone＇　can　be　improved　by　using　class-specific　matrices　and　gap　penalties.　The　substitution　pattern　differences　between　different　protein　classes　do　improve　the　performances　of　the　three　class-specific　matrices.　Analyzing　amino　acids　substitution　pattern　based　on　different　matrices　also　suggests　important　information　for　protein　sequence　design.　©　2008　IEEE.