The cellular physiology of signal transducer and activator of transcription protein family (STAT) transcription factors includes activation by Tyr-phosphorylation (PY) in cytokine and growth factor receptor complexes at the level of plasma membrane rafts, subsequent cytoplasmic transit and nuclear import, and transcriptional regulation of target genes, followed by dephosphorylation and export back to the cytoplasm. The ubiquitous protein tyrosine phosphatase (PTP) called "T-cell protein tyrosine phosphatase" has been reported to mediate Tyr-dephosphorylation of both interferon-gamma (IFN-gamma)-induced PY-STAT1 and interkleukin-6 (IL-6)-induced PY-STAT3 in some cell lines. To test whether the same PTP regulated both PY-STAT1 and PY-STAT3 in human hepatocytes we used orthovanadate (VO(4); 0.01-1.0mM) as a PTP-inhibitory probe and evaluated the kinetics of PY-STAT3 and PY-STAT1 accumulation, nuclear trafficking, and dephosphorylation following cytokine (IL-6 or IFN-gamma) stimulation of Hep3B cells. As evaluated using DNA binding or Western blotting assays, in IL-6-treated hepatocytes VO(4) had a modest enhancing effect on peak levels of cytoplasmic and nuclear PY-STAT3 reached by 1h and on their subsequent decline. In contrast, in the same cells and at the same time, VO(4) caused a marked and continuing increase in cytoplasmic and nuclear levels of PY-STAT1 which, by 4h, were 5- to 10-fold higher than peak levels reached in VO(4)-free, IL-6-treated cells. Prolonged treatment of cells with VO(4) alone (for 4-8h) replicated this markedly selective enhancement of PY-STAT1 levels. Consistent with this selectivity, shorter term VO(4) treatment (1-2h) markedly increased PY-STAT1 levels in all cellular compartments of IFN-gamma-treated cells by >10-fold. The unexpected selectivity in the effects of VO(4) on PY-STAT1 compared to that on PY-STAT3 levels in Hep3B cells suggests that, at least in these hepatocytes, the regulation of PY-STAT1 and PY-STAT3 likely involves distinct protein tyrosine phosphatase mechanisms.