We have previously isolated an agonist-regulated actin filament capping activity, called aginactin, that is associated with a 70-kDa protein (Sauterer, R. A., Eddy, R. J., Hall, A. L., and Coneelis, J. S. (1991) J. Biol. Chem. 266, 24533-24539). A 2.0-kilobase clone isolated from a Dictyostelium lambda gt11 cDNA library screened with affinity-purified aginactin antibodies displays an overall sequence identity of 73% to the 70-kDa heat shock cognate protein, Hsc70, from various species. Aginactin capping activity and the 70-kDa protein bind to ATP-agarose columns and are quantitatively depleted from the load, indicating that an Hsc70 is associated with aginactin activity. Moderate stringency Southern blots indicate the presence of no fewer than six Hsc70-related sequences. Immunofluorescent staining of vegetative Dictyostelium AX3 cells with aginactin antibodies reveals a colocalization of aginactin-associated Hsc70 in F-actin-rich regions of the cell cortex and cell protrusions. Nuclei and organelles lacked positive staining indicating that the aginactin-associated Hsc70 is cytosolic. The levels of cytoskeletal-associated Hsc70 correlate with the loss of barbed end capping activity following cAMP stimulation, suggesting that the uncapping of barbed filament ends through an Hsc70-associated process may account for the increase in nucleation activity observed at 5 s following agonist stimulation.