Previous studies have shown that a 20-kDa protein enhances production of the insecticidal CytA and CryIVA proteins of Bacillus thuringiensis in Eschel ichin coli as well as CytA production and crystal formation in B. thuringiensis. To determine whether the 20-kDa protein could enhance CryIVD production, an expression vector was constructed with the 20-kDa open-reading frame under control of cryIA(c) promoters and the a cryIVD gene under control of its own promoter. Acrystalliferous cells of B. thuringiensis transformed with this plasmid, designated pWF53, produced large bitrapezoidal CryIVD crystals that averaged 1.3 x 0.92 x 0.31 mu m, approximately fivefold larger in volume than wild-type CryIVD crystals, and 1.7 fold the volume of crystals produced using the cryIVD operon, which contains the cryIVD gene and the gene for the 20-kDa protein. These results demonstrate that the 20-kDa protein significantly improves net synthesis of CryIVD and promotes CryIVD crystal formation. Improved production of proteins as diverse as CryIVD and CytA by the 20-kDa protein indicates this protein may be useful in facilitating the production of other proteins.