Given the high similarity in genome content and organization between Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and Agrotis segetum nucleopolyhedrovirus (AgseNPV), as well as the high percentages of similarity found between their 30 core genes, the specificity of these NPVs was analysed for the respective insect hosts, S. exigua and A. segetum. The LD(50) for AgseNPV in second-instar A. segetum larvae was 83 occlusion bodies per larva and the LT(50) was 8.1 days. AgseNPV was orally infectious for S. exigua, but the LD(50) was 10 000-fold higher than for SeMNPV. SeMNPV was not infectious for A. segetum larvae when administered orally, but an infection was established by injection into the haemocoel. Bypassing midgut entry by intrahaemocoelic inoculation suggested that the midgut is the major barrier in A. segetum larvae for infection by SeMNPV. Delayed-early genes of SeMNPV are expressed in the midgut of A. segetum larvae after oral infections, indicating that the virus is able to enter midgut epithelial cells and that it proceeds through the first phases of the infection process. The possible mechanisms of A. segetum resistance to SeMNPV in per os infections are discussed.