Tyrosine phosphorylation in response to cytokine stimulation of cells is believed to be required for the nuclear translocation of cytoplasmic STAT proteins (signal transducers and activators of transcription). In this study we examined the nucleocytoplasmic distribution of STAT1 and STAT3 in transformed cell lines and primary cells prior to stimulation with cytokines. It was found that both STAT1 and STAT3 are constitutively nuclear in resting cells. Moreover, the extent of nuclear presence of both proteins differed in a cell type-specific mode as revealed by immunocytochemistry and confocal microscopy. We investigated whether varying degrees of tyrosine phosphorylation could account for these differences. The results show that depletion of type I interferons from culture medium with blocking antibodies did not influence the STAT1 distribution in unstimulated cells. In addition, blocking tyrosine kinase activity with staurosporine also did not influence the nucleocytoplasmic STAT1 distribution in resting cells. Nuclear extracts from unstimulated HeLa-S3 cells, which are demonstrated to be exceptionally high in the nuclear concentration of STAT1, did not contain detectable quantities of tyrosine-phosphorylated STAT1. In addition, the nucleocytoplasmic distribution of a STAT1 mutant which can no longer be phosphorylated or dimerize did not differ from wild-type protein. Thus, these data indicate that tyrosine phosphorylation of STATs does not constitute a mandatory requirement for the nuclear presence of these transcription factors.