Integrin-mediated tumor cell adhesion to type IV collagen is believed to play a role in the invasion of basement membrane proteins and the subsequent metastatic process. The cellular protein CAR (cell adhesion regulator) has been proposed to influence integrin-mediated binding to extracellular matrix proteins, including basement membrane (type IV) collagen. Three analogs of the CAR138-142 have been tested for activity. The first contains the 138-142 sequence (CAR138-142, Val-Glu-Ile-Leu-Tyr-NH2), the second contains the 138-142 sequence with a phosphorylated Tyr [pCAR138-142, Val-Glu-Ile-Leu-Tyr(PO3H2)-NH2], and the third contains the reversed 138- 142 sequence (rCAR138-142, Tyr-Leu-Ile-Glu-Val-NH2). When added extracellularly, none of the analogs had a significant affect on cell adhesion to type IV collagen. Using a novel reversible cell permeabilization method, we found that intracellular incorporation of both CAR138-142 and pCAR138-142 resulted in inhibition of cell adhesion in a dose- dependent fashion. The IC50 values were ~90 and ~10 μM for CAR138- 142 and pCAR138-142, respectively. Intracellular incorporation of the rCAR138-142 peptide had no affect on cecil adhesion. Fluorescence microscopy of a fluorescein-labeled CAR138-142 peptide revealed that the reversible permeabilization procedure resulted in the peptides crossing the cell membrane. Affinity chromatography of melanoma cell lysates with pCAR138-142 or rCAR138-142 attached to a solid support of magnetic beads suggested that one protein was bound uniquely by pCAR138- 142. Immunoprecipitation analysis identified vinculin, a protein associated with the actin cytoskeleton, as the protein specifically bound by pCAR138-142. Immunoprecipitation with pp125(FAK)- or β1-integrin- derived mAbs gave negative results. Our study suggests that a possible therapeutic approach for inhibition of melanoma cell adhesion adhesion to extracellular matrix proteins is the use of CAR peptide analogs intracellularly.