To identify novel cry1-type crystal protein genes, 100 Bacillus thuringiensis (Bt) isolates were selected on the basis of their toxicity against lepidopteran insect larvae. For rapid search of a large quantity of novel genes simultaneously, Bt isolates were randomly divided into 2 groups including 50 isolates each. About 2.4 kb PCR fragments were amplified from each group using universal oligonucleotide primers, ATG1-F and N400-R, designed to probe the toxic fragment regions of all known and possible cry1-type genes. Restriction fragment length polymorphism (RFLP) analysis revealed 3 distinct patterns in each group. One of them, A32 clone showed 87% and 91% of nucleotide sequence and deduced amino acid sequence similarities with the cry1Ea gene. Interestingly, nucleotide sequence analysis of A32 cry gene suggested that this gene might have resulted from nucleotide rearrangement between cry1Ac and cry1Ea genes.