The venom of Microplitis demolitor consists of a mixture of proteins. On native PAGE gels three major proteins designated a, b, and g were detected, while on SDS-PAGE gels two major proteins of M(r) 64.5 and 30.8 kD and several minor proteins were detected. No proteins smaller than M(r) 30.8 kD were present. Murine monoclonal antibodies were generated against different venom components. Analysis by Western blot of venom proteins separated on native and SDS-PAGE gels confirmed that antibodies from seven hybridoma lines recognized venom components. Two of the seven hybridoma lines reacted specifically with protein g on native PAGE gels and the M(r) 30.8 k protein on SDS-PAGE gels, while four other lines cross-reacted with these and other venom proteins. The final hybridoma line reacted with protein a when venom was separated on native PAGE gels and an array of proteins when venom was separated on SDS-PAGE gels. Using an enzyme-immunoassay and specific monoclonal antibodies, M. demolitor females were estimated to inject 0.02-0.05 venom gland reservoir equivalents into its host, Pseudoplusia includens, at oviposition. Venom proteins persisted in host hemolymph for 6-12 h before dropping to undetectable levels.