Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with Protein kinase C (PKC). We have studied the interaction of calponin with PKC and with the low molecular weight heat shock protein HSP27, in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blotted with antibodies to HSP27 and to PKC. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nucleotides of calponin cDNA (nt 351-972 corresponding to aa 92-229), was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-Calponin fusion protein with recombinant PKC in vitro. Western blots of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a co-elution of GST-Calponin with PKC, indicating a direct association of GST-Calponin with PKC. These data suggest that there is a direct association of translocated calponin and PKC in the membrane and a role for the complex calponin- PKC-HSP27, in contraction of colonic smooth muscle cells.