We have constructed a modified Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to express the green fluorescent protein (GFP) under the polyhedrin promoter and used it to study the infection process of AcMNPV in Trichoplusia ni larvae. T. ni larvae that ingested the virus showed localized expression of GFP in the midgut epithelial cells and the hemocytes at 12 h post infection (hpi). The presence of GFP-related fluorescence in the midgut columnar cells indicated that the virus was not only replicating, but also synthesizing the late viral proteins. Studies using the transmission electron microscope showed that the virus infected the midgut columnar cells. At the same time a proportion of the parental virus travelled through the midgut epithelial layer, possibly utilizing the plasma membrane reticular system, entered the hemocoel and infected the hemocytes. This resulted in the simultaneous infection of the midgut epithelial cells and the hemocytes. Subsequently, the budded virus (BV) released from the infected hemocytes into the hemolymph caused secondary infection within the tracheal epithelial cells. The virus then rapidly spread through the tracheal system allowing the infection of a variety of other tissues such as the epidermis and the fat body.