A novel Cre-lox system was used to construct an adenovirus encoding kappa-bungarotoxin (kappa-Bgt), modified to be secreted by attachment of a bovine prolactin signal sequence at the N-terminus of the toxin. Western blot of medium from HEK-293 cells infected with the virus demonstrated that recombinant kappa-Bgt (R-kappa-Bgt) was secreted. The biological activity of the secreted R-kappa-Bgt was investigated in Xenopus oocytes that expressed neuronal nicotinic acetylcholine receptor (nAChR) subtypes alpha3beta2 and alpha2beta2. The recombinant toxin inhibited the response of alpha3beta2 type AChRs to ACh, but did not inhibit the response of alpha2beta2 type AChRs. These data demonstrated that the recombinant adenovirus directs the secretion of biologically active kappa-Bgt from a mammalian cell line. Because adenovirus can be used to infect post-mitotic cells, recombinant adenoviruses encoding biologically active peptides may be of use as delivery vehicles for in vivo experiments where repeated application of the purified peptide is unfeasible.