A insertional mutant library of Bacillus thuringiensis strain YBT-1518 was constructed with a thermo-sensitive plasmid pIC333 carrying Mini-Tn10.A mutant (named MA1) defective in germination response to L-alanine was isolated from the library by detecting with phase-contract microscopy and D600 nm.Further studies indicate that MA1 was defective in germination response to other amino acid (such as L- phenylalanine,L-valine and L-leucine) and sugars (such as glucose,sucrose and fructose),different from other reported mutants.Results indicate that MA1 is a new type defective in spore germination.