Bacillus thuringiensis subsp. kurstaki produces a proteinaceous crystalline inclusion that is toxic for lepidopteran larvae. There are several size classes of plasmids in this organism and the presence of one or more has been correlated with production of this protein, defined as a protoxin. DNA fragments of B. thuringiensis subsp. kurstaki, obtained by EcoRI digestion, were cloned into the vector Charon 4A. Recombinant phage were screened immunologically for the production of protoxin. Cells infected with one phage, C4K6c, produced antigen that was the same size as the protoxin and was toxic to Manduca sexta larvae. A 4.6-kilobase-pair (kbp) EcoRI fragment from C4K6c was subcloned into pBR328 and in both orientations in pHV33. Both Escherichia coli and Bacillus subtilis containing these recombinant plasmids produced antigen that crossreacted with antibody directed against the protoxin. The various sized plasmids of B. thuringiensis were purified and only an EcoRI fragment from the 45-kbp plasmid hybridized to phage C4K6c. One of the pHV33 subclones, pSM36, hybridized to the same size EcoRI/HindIII restriction fragments from plasmid or chromosomal DNA. The cloned EcoRI fragment contained a 0.9-kbp Pvu II fragment that was also present in chromosomal but not in plasmid digests. The original clone was therefore of chromosomal origin, although very similar or identical protoxin genes were present in both the 45-kbp plasmid and the chromosome. Several acrystalliferous nontoxic mutants have been isolated that lacked the 45-kbp plasmid and in some cases all plasmids. All of the mutants contained the chromosomal gene but did not produce protoxin antigen.