The hypothesis that was tested is PKC mediates the phosphorylation of glycogen synthetase kinase 3 (GSK3) and that the GSK3 inhibition modulates the response to tumor necrosis factor- (TNF) in rat pulmonary microvessel endothelial cells (PMEC). PMEC were treated with TNF for 4.0 hr (100 ng/ml) or vehicle. First, to assess the role of PKC in the phosphorylation of GSK3 (i.e., an indicator of GSK3-inhibition), PMEC were pre-treated with: (1) nonsense RNA-PKC, (2) siRNA-PKC, and (3) the PKC inhibitor Go6983. In the nonsense RNA-PKC+TNF and TNF groups, there were increased phosphorylated-GSK3-Ser⁹ which did not occur in the Go6983+TNF group. In the TNF groups, there was a significant correlation between PKC protein and phosphorylated-GSK3-Ser⁹ which did not occur in the groups without TNF. Second, to assess the role of GSK3 in -catenin activity, PMEC were pre-treated with: (1) wild type (w) GSK3-plasmid to enhance GSK3 activity, (2) kinase dead (kd)-GSK3-plasmid, and (3) the GSK3 inhibitor SB216763. In the TNF group, there was increased un-phosphorylated--catenin-Ser ³⁷/ ³³ compared to the Control group. In the GSK3-inhibited groups (i.e, SB216763 and kdGSK3) ± TNF, the un-phosphorylated--catenin-Ser ³⁷/ ³³ was similar to the TNF group. In the GSK3-enhanced group ± TNF, the un-phosphorylated--catenin-Ser ³⁷/³³ was similar to the Control. Finally, RPMEC were also treated with TOPflash, a -catenin-dependent-promoter luciferase reporter, or the mutant construct FOPflash, two days prior to treatment with TNF. In the TNF group, there was an increased TOPflash/FOPflash activity ratio compared to the Control group. In the GSK3-inhibited groups (i.e, SB216763 and kdGSK3) ± TNF, the TOPflash/FOPflash activity ratio was similar to the TNF group. In the GSK3-enhanced group ± TNF, the TOPflash/FOPflash activity ratio were similar to the Control. The data indicate that TNF-induces endothelial activation that is modulated by a PKC-dependent inhibition of GSK3.