We previously reported that cAMP induces Ca²⁺-desensitization in mouse tail arteries by PKA-mediated activation of myosin phosphatase, MLCP. Here we investigated whether cAMP-mediated Ca²⁺-sensitization additionally involves activation of Epac and its downstream target Rac1. Submaximally contracted (pCa 6.1) a-toxin permeabilized mouse-tail arteries were incubated with the specific activator of PKA (6-Bnz-cAMP, Bnz) or Epac (8-pCPT-O-Me-cAMP, pCPT) in a concentration dependent manner with and without the Rac1 inhibitor, NSC 23766 (400 mM). Phosphorylation of MLC₂₀ at S19 and the regulatory subunit of MLCP, MYPT1 at T696 & T863 were determined by Western blot analysis using phosphospecific antibodies. Both cAMP analogues relaxed the preparations in a concentration-dependent manner at constant pCa 6.1. At equipotent concentrations (1 mM Bnz and 30 mM pCPT) relaxation amounted to 65% and 58% respectively which was associated with a decrease in MLC₂₀ phosphorylation by 32% (Bnz) and 23% (pCPT). Addition of NSC at the plateau of the pCa 6.1 induced contraction increased force which was associated with a 2-fold increase in MLC₂₀ phosphorylation without increasing phosphorylation of MYPT1 suggesting that NSC leads to disinhibition of MLCK. Pretreatment with NSC neither affected Bnz induced relaxation nor MLC₂₀ and MYPT1 dephosphorylation whereas it attenuated cCPT induced relaxation and MLC₂₀ dephosphorylation by ~50%. Our results suggest that Epac-induced Ca²⁺-desensitization is partially mediated via Rac1-dependent inhibition of MLCK. This work also suggests that the down-stream mechanisms by which PKA and Epac decrease Ca²⁺-sensitivity are distinct.