Full-length (72K) and truncated (61K) CrylVD mosquitocidal proteins of Bacillus thuringiensis (Bt) were expressed in Spodoptera frugiperda cells and larvae of Trichoplusia ni using a baculovirus vector to investigate the role of CrylVD peptides in toxicity as well as to evaluate further the baculovirus/lepidopteran system for expressing Bt proteins. The crylVD genes were inserted into the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) under control of the polyhedrin promoter by recombination in S. frugiperda cells between a transfer vector carrying the Bt genes and vDA26Z, a recombinant AcMNPV carrying the Escherichia coli fl-galactosidase gene under control of the DA26 promoter. Recombinant AcMNPVs carrying the genes were detected as blue occlusion body-negative plaques in monolayers of S. frugiperda cells grown in the presence of X-Gal. Infection of S. frugiperda cells and T. ni larvae with plaque-purified recombinant virus, expressing either the full-length or truncated CrylVD protein, resulted in the synthesis of proteins of the expected size, as confirmed by immunoblot analyses, and their crystallization into cuboidal inclusions in the cytoplasm. Infected cells and purified inclusions from the virus (AcCrylVD) expressing the full-length protein were highly toxic to mosquito larvae, but similar preparations from the virus (AcCrylVD-C) expressing the truncated protein with a 9-6K deletion at the N terminus were non-toxic. Proteolysis with trypsin of CrylVD proteins produced by Bt and the recombinant AcMNPVs yielded peptides corresponding in size, showing that synthesis of mosquitocidal Bt proteins in lepidopteran cells occurred. The lack of toxicity of the truncated CryIVD protein, which like the toxic fulllength protein yielded a 34K protein on proteolysis that has been implicated in toxicity, indicates that by itself this protein is non-toxic. These results demonstrate the utility of the baculovirus system for expression of mosquitocidal Bt proteins and for investigation of their mode of action.