Using monolayers of intestinal Caco-2 cells, we have been investigating cellular and barrier damage by oxidants, and protection by growth factor-triggered signaling pathways. We reported that activation of NF-B, an inflammatory mediator, is required for oxidative disruption and that EGF protects against this injury. PKC activation, especially activation of the classical 1 isoform, is key to monolayer barrier integrity. To investigate the molecular processes underlying EGF protection against oxidant disruption, we hypothesized that EGF-induced activation of PKC-1 prevents oxidant-induced activation of NF-B and the consequences of NF-B activation, namely F-actin disassembly and barrier dysfunction. Methods: Monolayers of wild type (WT) Caco-2 cells were exposed to oxidant (H₂O₂) ±EGF or modulators. In separate studies, we used Caco-2 clones created by stable transfection of varying levels of cDNA to either over-express or under-express PKC-1. These clones were then pretreated with EGF or PKC modulators (DAG analog, OAG) prior to oxidant. Results: {A} Relative to oxidant-exposed WT type cells, pretreatment with EGF protected monolayers by: 1) increasing native PKC-1 activity; 2) suppressing 6 variables related to NF-B activation [p50/p65 subunits nuclear translocation, NF-B subunits activity, I-B- degradation/phosphorylation]; 3) enhancing stable actin (increased F-actin, decreased G-actin); 4) stabilizing the architecture of actin cytoskeleton; and 5) preventing barrier hyperpermeability. {B} Transfection of cells to stably over-express PKC-1 (~3.1 fold) also led to protective conditions; these clones were protected by low, previously non-protective doses of EGF or OAG. In these clones, we also found protection against NF-B activation, I-B degradation, actin instability, and barrier hyperpermeability. PKC-1 over-expression induced stabilization of I-B was potentiated by EGF or OAG. In transfected clones low doses of modulators led to increases in PKC-1 in the particulate fractions, indicating activation -1 isoform. {C} Stably inhibiting native PKC-1 (-99%) substantially inhibited all measures of EGF-mediated protection against NF-B activation and F-actin dysfunction. Conclusions: (1) EGF-mediated protection against oxidant disruption of the intestinal barrier function requires the activation of PKC-1 isoform and suppression of NF-B; (2) The molecular event underlying this unique biologic effect of the classical PKC isoform -1 appears to involve inhibition of phosphorylation and increases in stabilization of I-B; (3) The ability to inhibit the dynamics of NF-B and F-actin cytoskeletal disassembly is a novel mechanism not previously attributed to the classical subfamily of PKC isoforms.