Agonist-induced activation of RhoA/Rho kinase pathway results in inhibition of myosin phosphatase and maintenance of MLC₂₀ phosphorylation. We have shown that RhoA/ROCKII translocate and associate with HSP27 in the particulate fraction. We hypothesize that inhibition of MYPT requires its association with HSP27 in the particulate fraction. Further, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to a cross talk between RhoA and PKC. Presently we have examined the cross talk between RhoA and PKC in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced: 1) sustained phosphorylation of PKC, CPI-17 and of MYPT, 2) Increase in the association of Phospho-MYPT with HSP27 in the particulate fraction, 3) Decrease in myosin phosphatase activity (66.21 ± 3.52 and 42.19 ± 3.85 percent in nMoles/ml of lysate at 30 s and 4 min), 4) Increase in PKC activities (298.12 ± 46.60 and 290.59 ± 22.07 percent at 30s and 4 min). Inhibition of RhoA/ROCKII by Y27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs by Calphostin C or selective inhibition of PKC by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that: 1) Acetylcholine induces activation of both RhoA and/or PKC pathways suggesting a cross talk between RhoA and PKC resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity and maintaining MLC phosphorylation. 2) Phosphorylated MYPT associated with HSP27 and translocated to the particulate fraction suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus, both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of MLC₂₀.