Both GH and the GH receptor have been reported to undergo rapid nuclear translocation. Janus kinases (JAK) 1 and 2 have been implicated in GH receptor signaling, and both of these kinases are phosphorylated by GH stimulation. In this report, we have investigated the subcellular distribution of JAK1 and JAK2. Both JAK1 and JAK2 exhibit a nucleocytoplasmic distribution by immunocytochemistry in unstimulated serum deprived CHO cells stably transfected with rat GH receptor complementary DNA (cDNA). The nucleocytoplasmic localization of JAK2 was verified by immunogold electron microscopy in both rat liver hepatocytes and CHO cells stably transfected with rat GH receptor cDNA. Nucleocytoplasmic localization of JAK2 was also verified by transient tranfection of CHO cells with a Haemophilus influenzae haemagglutinin (HA) epitope tagged JAK2 expression plasmid and subsequent localization of HA immunoreactivity. Western blot analysis of purified nuclear extracts revealed the presence of immunoreactive JAK1 at 130 kDa and immunoreactive JAK2 at 128 kDa. No change in the nuclear content of JAK1 or JAK2 was observed upon ligand stimulation of GH receptor cDNA transfected cells with 100 nM human GH for 5, 10, 15, 30, or 60 min. GH stimulation caused, however, the appearance of tyrosine phosphorylated 42- and 44-kDa proteins as well as tyrosine phosphorylated JAK2 in the nucleus. The constitutive nuclear localization of the Janus Kinases is suggestive of a novel nuclear role for JAK family members, in addition to their described cytosolic function and presents an interesting challenge to the subcellular site of hormone action.