Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKC in the membrane. In the present study, we have determined the domain of PKC involved in direct association with calponin. In vitro binding assay was carried out by incubating GST-calponin with His-tagged proteins of individual domains and different combinations of domains of PKC. Calponin was found to bind directly to the full length PKC. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4 and to C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these In vitro bindings mimic the in vivo associations, in vivo binding assay was performed by transfecting colonic smooth muscle cells with His-tagged proteins of individual domains and different combinations of domains of PKC. Co-immunoprecipitation of calponin with His-tagged truncated forms of PKC showed that C1-C2, C1-C2-C3, C2-C3 and C3-C4 did not associate with calponin. Calponin associated only with full length PKC and with C2-C3-C4 in cell sin the resting state, and this association increased upon stimulation with acetylcholine. These data suggests that calponin bound to fragment that may mimic the active form of PKC and that the functional association of PKC with calponin require both C2 and C4 domains during contraction of colonic smooth muscle cells.