We have previously shown that the stress related hormone, urocortin 1 (UCN-1) in a cAMP dependent manner decreased Ca2+-sensitivity of vascular smooth muscle contraction and MLC20 phosphorylation by increasing the activity of myosin light chain phosphatase, MLCP associated with dephosphorylation of MYPT1 at T696/T853 (Lubomirov et al., 2006). Here we show, that Urocortin / cAMP-mediated dephosphorylation of MYPT1 can be ascribed to both phosphorylation of S695 known to prevent subsequent inhibitory phosphorylation at T696 and a decrease in the amount of active RhoA. Using cAMP analogues which activate either relatively specific PKA (6-bnz-cAMP) or EPACs (8-pCPT-cAMP) we tested whether so called exchange proteins directly activated by cAMP (EPACs, Bos, 2003) contribute to activation of MLCP. Ca2+-sensitivity and MYPT1 phosphorylation in α-toxin permeabilized mouse tail arteries is decreased by both cAMP analogues to a similar extent albeit with different potencies. Western blots with phosphospecific antibodies against P-Ser695 of MYPT1 revealed that as expected only 6-bnz-cAMP but not 8-pCPT-cAMP increased phosphorylation of MYPT1-S695. 6-bnz-cAMP like urocortin decreased active RhoA-GTP by 80% as determined by the Rhotekin-pulldown assay. However, surprisingly, 8-pCPT-cAMP decreased RhoA-GTP only by ~40% (n=4, p<0.05). In conclusion, cAMP decreases Ca2+-sensitivity mediated by disinhibition of MLCP in a PKA-dependent and PKA-independent manner. While both pathways converge at the level of MYP1 and decrease phosphorylation of T696 and T853, whereas the upstream mechanisms only partially overlap.