The acquisition of an invasive and metastatic phenotype is accompanied by profound alterations of intracellular mechanisms controlling cell movement. Analysis of quantitative parameters of cell motility in cancer cells may help in the identification of intracellular signaling events determining invasion and metastasis. Here we developed a novel procedure of quantification of cell motility based on time-lapse video microscopy and digital image analysis. Three kinetic parameters, including area change, plasma membrane remodeling, and speed of linear movement, are quantified and combined in one single, time-normalized value we defined motility score (MS). Through calculation of the MS for various human pancreatic adenocarcinoma cell subclones, we identified clones characterized by low or high spontaneous motility in vitro. Analysis of the signaling mechanisms involved in the regulation of pancreatic adenocarcinoma cell motility showed that the atypical ζ isozyme of the serine-threonine protein kinase C (PKC) plays a critical role in maintaining a high MS in motile subclones, as demonstrated by the inhibitory effect of cell permeable peptides with sequence corresponding to the pseudosubstrate inhibitory region of the atypical ζ PKC. Other PKC isozymes, either classic or novel, seem not involved. Furthermore, biochemical analysis showed that in motile cells, ζ PKC is constitutively associated with the plasma membrane, whereas in nonmotile cells, ζ PKC is totally excluded from the plasma membrane. These data suggest that the disregulation of the function of atypical ζ PKC might be involved in the acquisition of an invasive and metastatic phenotype in pancreatic adenocarcinoma cells.