CHO cells spread out on fibronectin-coated plastic were perforated with the bacterial toxin alveolysin. This treatment preserves the integrity of the cells but opens large and stable hydrophilic pores on the plasma membrane. With these semi-intact cells, it has been possible to have a direct access to adhesion plaques. Furthermore, with this procedure one can determine the distribution of a single intracellular protein between membrane-associated and cytosolic pools. The introduction within the perforated cells of polyclonal antibodies raised against talin induced the detachment of the cells. This provides direct evidence that talin is required to stabilize the adhesion plaques. Furthermore, immunoprecipitation of talin in the membrane-associated and cytosolic fractions indicated that the membrane-associated talin was cleaved and reduced to a stable 200-kDa fragment. Conversely, soluble talin remained intact. This 200-kDa fragment was similar to the proteolytic fragment produced from talin by calpain II. Analysis of whole cell extracts and pulse chase experiments indicated that this proteolysis also occurred in vivo, although to a smaller extent. The kinetics of the limited proteolytic cleavage of talin suggested that the 200-kDa fragment was first produced within focal adhesion, and secondarily released into the cytosol.