Midgut epithelial ceils were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several ceils lines were obtained from these P. unipuncta prima3 cultures, and they have been subcultured and maintained for over 24 too. The three major midgut cell types were present in the cultures, including stern (regenerative), columnal, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem ceils were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells evely 7-8 wk. After approximately six passages, the cell density in Tflasks appeared to be somewhat constant, reaching 103-104 cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these ceils with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.