α‐latrotoxin, α‐latroinsectotoxin and the low‐molecular‐mass protein from black widow spider venom were synthesised in insect cells using the baculovirus expression system. SDS/PAGE analysis of recombinant‐virus‐infected cells revealed novel proteins that migrated with sizes similar to those of the neurotoxins from spider venom. The identities of these proteins as α‐latrotoxin, α‐latroinsectotoxin or the low‐molecular‐mass protein were confirmed by immunoblot analysis of infected cells with anti‐(α‐latrotoxin), anti‐(α‐latroinsectotoxin) or anti‐(low‐molecular‐mass protein) IgG. Neither the low‐molecular‐mass protein nor α‐latrotoxin were toxic upon injection into Trichoplusia ni larvae or upon virus‐derived synthesis directly in the cytoplasm of the target tissue. Analysis of the biological activity of the recombinant virus encoding α‐latroinsectotoxin, however, revealed a strong toxic effect on the T. ni larvae. These data indicate that the toxic effect of the native insectotoxin may be promoted by the α‐latroinsectotoxin subunit alone and provides evidence that the mechanism of action of α‐latroinsectotoxin may be mediated by internalisation of part of the neurotoxin α‐subunit molecule.