Influenza virus nucleoprotein (NP) plays a major role in the nucleus during virus replication, and is a mediator of viral ribonucleoprotein nuclear import during entry. NP is localized primarily in the nucleus, but can undergo nucleo-cytoplasmic shuttling in heterokaryons (Whittaker et al., 1996a. J. Virol. 70, p. 2743). We have studied NP localization using a stable cell line (3PNP-4) that expresses NP. Intracellular localization of NP was markedly affected by the density of the cell monolayer. It was nuclear in cells grown in sparse culture, but cytoplasmic in dense culture. In phorbol ester-stimulated cells NP was cytoplasmic, but relocalized to the nucleus after treatment with a protein kinase inhibitor. Cell density and phosphorylation-dependent localization of NP appeared to be independent of cell type. Our data suggest that a phosphorylation event is needed either for nuclear export, or to regulate retention of NP in the nucleus, and that regulation may be mediated by kinases activated by cell-cell contact.