Direct binding of non-smooth muscle F-actin and the C2-like domain of PKC. (C2-like domain) is involved in hormone-mediated activation of epithelial NaK2Cl cotransport (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid C2-like domain. Truncations of the C2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His6-tagged peptides were expressed in bacteria, purified, and analyzed using a Coomassie blue stain for predicted size and either a 6xHis Protein Tag Stain or India His6 Probe for expression of the His6 tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC-with non-smooth muscle F-actin. Peptides from the N-terminal region, but not the C-terminal region, of the C2-like domain blocked binding of activated PKC- to F-actin. The C2-like domain and three N-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding with IC₅₀ values ranging from 1.2 nmole to 2.2 nmole (6 µM to 11 µM). N-terminal C2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal N-terminus of the C2-like domain encodes a 1-sheet region. The amino acid sequence of the actin binding domain is distinct from actin binding domains in other PKC isotypes and actin binding proteins. Our results indicate that F-actin likely binds to the 1-sheet region of the C2-like domain in airway epithelial cells.