Using monolayers of intestinal Caco-2 cells, we previously showed that assembly of the cytoskeleton (e.g., microtubules) is required for monolayer stability. However, the molecular mechanisms underlying alterations to cytoskeletal assembly remain poorly understood. Because the theta () isoform of PKC, a member of novel subfamily of PKC, is present in our wild type intestinal cells, we tested the hypothesis that PKC- isoform activity is required for changes in the dynamics of cytoskeleton in monolayers of intestinal epithelium. Methods: Using targeted molecular interventions, we have created the first, multiple sets of GI cell clones transfected with varying levels (1-5 µg) of cDNA to stably inhibit native PKC-(anti-sense, dominant negative) or to express its activity (sense). We studied both transfected and wild type (WT) Caco-2 cells. We monitored the following outcomes: monolayer stability {clearance of different size probes, FS and FD4}; cytoskeleton {microtubules and occludin by laser confocal microscopy}; PKC-theta intracellular distribution (cytosolic, membrane & cytoskeletal fractions) {immunoblotting, immunoprecipitation analysis}; PKC- isoform activity {in vitro kinase assay}; polymerized & monomeric tubulin pools {50 kDa structural protein of microtubules, PAGE}, and tubulin phosphorylation {PAGE}. Results: {A} Relative to WT cells exposed to vehicle, anti-sense transfected cells stably under-expressing the PKC-theta (-99.5%) showed monolayer injury as indicated by: [1] decreased native PKC-theta activity, [2] reduced tubulin phosphorylation, [3] increased tubulin disassembly (decreased polymerized pool, increased monomeric pool), [4] reduced architectural integrity of the microtubule cytoskeleton, [5] reduced architectural stability of the occludin, and [6] increased monolayer instability (hyperpermeability). In these anti-sense clones PKC- was substantially reduced in the particulate (membrane + cytoskeletal) fractions, indicating its inactivation. In WT cells, on the other hand, PKC- (82 kDa) was constitutively active and co-associated with tubulin (50 kDa), forming an endogenous PKC-/tubulin complex. {B} In a 2^nd series of studies, dominant negative transfection to stably inhibit the activity of native PKC- (inactivating endogenous ) led to similar destabilizing effects on monolayers, including tubulin hypo-phosphorylation, tubulin depolymerization, microtubule architectural instability and hyperpermeability. {C} In a 3^rd series of studies, stable over-expression of the PKC- (+2.0 fold) led to a mostly cytosolic distribution of this isoform (<10% in the membrane and cytoskeletal fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased monolayer permeability. Conclusions: We show several original concepts: (1) PKC- isoform activity appears to be required for changes in the dynamics of cytoskeleton in monolayers of intestinal cells. (2) The molecular event underlying this new biologic effect of the PKC isoform - appears to involve changes in the phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the assembly of cytoskeleton is a unique function not previously attributed to the novel subfamily of PKC isoforms in cells.