Bacillus thuringiensis accumulates, primarily during sporulation, large quantities of insecticidal protoxins which are deposited as crystalline, intracellular inclusions. Most subspecies contain several plasmid-encoded cry genes, each of which has a unique specificity. The overall toxicity profile of a subspecies depends not only on the array of cry genes present but also on the relative expression of the genes. In general, transcription depends on sporulation-specific sigma factors, but little is known about regulation of expression of the individual genes. In order to determine whether expression of a particular cry gene varies in different subspecies, lacZ fusions to the cry promoters of two protoxin genes (cry1 class) were constructed. Protoxin accumulation and mRNA contents were also measured by performing immunoblotting and Northern analyses, respectively. The expression of a cry1Ab-lacZ fusion, but not the expression of a cry1C-lacZ fusion, was three to four times lower in B. thuringiensis subsp. aizawai strains than in B. thuringiensis subsp. kurstaki or B. thuringiensis subsp. tolworthi. Also, the Cry1Ab antigen and steady-state mRNA contents of B. thuringiensis subsp. aizawai were lower. The regulation of the genes must involve regions upstream of the promoters which are unique to each cry gene since (i) mutations in the upstream region of the cry1Ab gene resulted in enhanced expression in B. thuringiensis subsp. aizawai and (ii) no differences were found when the lacZ fusions contained the cry1Ab promoters but no upstream sequences. The capacity to regulate each of the protoxin genes must be a factor in the overall protoxin composition of a subspecies and thus its toxicity profile.