The shuttle vector pHT3101 and its derivative pHT408, bearing a copy of a cryIA(a) δ-endotoxin gene, were transferred into several Bacillus thuringiensis subspecies through phage CP-54Ber-mediated transduction, with frequencies ranging from 5 × 10-8 to 2 × 10-6 transductant per CFU, depending on the strain and on the plasmid. In Cry- and Cry+ native recipients, the introduction of the cryIA(a) gene resulted in the formation of large bipyramidal crystals that were active against the insect Plutella xylostella (order Lepidoptera). In both cases, high levels of gene expression were observed. Transductants displaying a dual specificity were constructed by using as recipients the new isolates LM63 and LM79, which have larvicidal activity against insects of the order Coleoptera. It was not possible, however, to introduce pHT7911 into B. thuringiensis subsp. entomocidus, aizawai, or israelensis by transduction. However, electrotransformation was successful, and transformants expressing the toxin gene cryIIIA, carried by pHT7911, were obtained. Again, high levels of expression of the cloned gene were observed. The results indicate that CP-54Ber-mediated transduction is a useful procedure for introducing cloned crystal protein genes into various B. thuringiensis recipients and thereby creating strains with new combinations of genes. Finally it was also shown that pHT3101 is a very good expression vector for the cloned δ-endotoxin genes in the different recipients.