A C-terminal region of human endothelial actin-binding protein-280 (ABP-280 or ABP, non-muscle filamin) was subcloned and efficiently expressed in a mammalian cells system as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis. As predicted by the aminoacid sequence, the fragment, a 79 kD peptide (residues 1671-2361, plus 3.9 kD from an N-terminal fusion peptide included in the expression plasmid), contained the two potential cAMP-dependent protein kinase (PKA) phosphorylation sites (serine 2152 and threonine 2336) predicted to be present in this region of the molecule. Incubation of cells in the presence of cAMP-elevating agents enhanced 32P uptake into the fragment. Site-directed mutagenesis analysis indicated that serine 2152 is the unique substrate in the C-terminal region of ABP for endogenously activated PKA. The functional implications of phosphorylation of this residue, which belongs to a serine-proline motif, are discussed in terms of the role of filamin in cytoskeleton reorganization.