Previous studies from this laboratory showed that agonist induced contraction of smooth muscle is associated with translocation of PKC and RhoA to the membrane, and that this interaction is due to a direct protein-protein interaction. In order to determine the domains of PKC involved in direct interaction with RhoA, His-tagged PKC proteins of individual domains and different combinations of domains PKC were used to carry out in vitro binding assay with fusion protein GST-RhoA. Coimmunoprecipitation was also performed by using smooth muscle cells transfected with truncated forms of PKC in this study. The data indicate that RhoA directly bound to full length PKC both in vitro(82.57±15.26% above control) and in transfected cells. RhoA bound to PKC(C1) (70.48±20.78% above control), PKC(C2) (72.26±29.96% above control) and PKC(C4) (90.58±26.79% above control), but not to PKC(C3) domain (0.64±5.18% above control) in vitro. RhoA bound to truncated forms of PKC, PKC(C2C3C4), PKC(C3C4) (94.09±12.13%, 85.10±16.16% above control respectively) in vitro and in transfected cells, but not to PKC(C1C2C3), PKC(C2C3) (0.47±1.26%, 7.45±10.76% above control respectively) both in vitro and in transfected cells. RhoA bound to PKC(C1C2) (60.78±13.78% above control) only in vitro, but not in transfected cells, PKC(C2C3C4) and PKC(C3C4) bound to RhoA well. It suggests that RhoA bound to fragments that may mimic the active form of PKC. The studies using cells transfected with truncated forms of PKC indicate that PKC(C1C2), PKC(C1C2C3) and PKC(C2C3) did not associate with RhoA. Only full length PKC, PKC(C2C3C4) and PKC(C3C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC with RhoA may require the C4 domain.