Urocortin (UCN1), a peptide related to corticotropin releasing factor, decreases Ca2+ -sensitivity of smooth muscle contraction by a cAMP dependent activation of SMPP-1M. We tested whether this is due to PKA-induced phosphorylation of MYPT at S695 or a PKA-independent activation of SMPP-1M. In permeabilized mouse tail arteries at pCa 6.1 UCN1 increased MYPT phosphorylation at S695 and decreased it at T696. The changes in phosphorylation were inhibited by Rp-8-CPT-cAMPS. MYPT-S695 phosphorylation was also increased by the specific PKA activator 6-Bnz-cAMP (Bnz) but not by pCPT-cAMP, an activitor of novel, non-PKA targets of cAMP (Epacs). However, both compounds decreased the phosphorylation of MYPT-T696. In permeabilized arteries preconstricted by pCa 6.1 both analogues decreased Ca2+ -sensitivity (pEC50 Bnz 6.6±0.03 and pCPT 4.8±0.02) with Rmax values of 77% and 62% respectively which were associated with a decrease in myosin light chain phosphorylation by 80±2.6 % (Bnz) and 70±8.9 % (pCPT). In conclusion our results suggest that UCN1 via cAMP activates SMPP-1M through a decrease in the inhibitory phosphorylation of MYPT at T696 by a PKA-dependent phosphorylation of S695 thereby preventing phosphorylation of T696 and a PKA- independent mechanism. We are currently investigating whether the latter is due to inactivation of the Rho / Rho-kinase pathway.