Cells exposed to phorbol 12-myristate 13-acetate (PMA) undergo a choreographed sequence of morphological changes. Some of these, including stimulation of membrane ruffles and the later appearance of stress fibers, rely on remodeling of the actin cytoskeleton. Although this process is poorly understood, it is important, because the same features are affected during oncogenic transformation. PMA also activates protein kinase C (PKC). Enzyme activation is followed by degradation. Either process might affect the remodeling of actin. The present studies determined whether any PKC isozymes were subject to degradation in tracheal epithelial cells by quantifying the amount of each isozyme present after PMA exposure. PKC-ε was the only isozyme to show declining content correlated with increased stress fiber accumulation. Stress fibers increased between 5 and 10 h, whereas PKC-ε declined to 38% of its starting value (95% confidence interval, 10–68%). The relationship could be fit by the function F(x) = 0.683 × exp[−0.841(x − 0.387)], where F is the frequency of fiber-containing cells and x is PKC-ε content. Fiber accumulation was further investigated after knockdown of PKC-ε with RNA interference and antisense oligodeoxynucleotide. Knockdown enhanced stress fibers in cells not yet exposed to PMA as well as the final frequency of fiber-containing cells after PMA exposure. With knockdown at both transcriptional and protein levels, ∼15% of the original content was predicted and achieved, as judged from real-time PCR and PKC-ε content measurements. The results suggest that PKC-ε negatively regulates stress fibers, either by directly turning over one of their components or by regulating an upstream step affecting fiber organization.