Contractile stimuli can sensitize myosin to Ca²⁺ by activating rhoA kinase (ROK) and protein kinase C (PKC) that inhibit MLC phosphatase (MLCP) activity. Relaxant stimuli, acting through PKA and PKG (cyclic nucleotide-dependent protein kinases), and pretreatment with contractile agents such as phenylephrine (PE), can desensitize myosin to Ca²⁺. Precisely how these stimuli cause Ca²⁺ desensitization remains to be determined. To test the hypothesis that PKA, PKG and PE-pretreatment signaling systems converge to cause relaxation by inhibition of ROK in intact, isolated tissues, we examined the effects of forskolin (FSK; PKA activation), 8-bromo-cGMP (8b-cGMP; PKG activation), and PE-pretreatment on KCl-induced force-maintenance in rabbit arteries, a response nearly completely dependent on ROK activation. PE-pretreatment and agents activating PKA and PKG caused Ca²⁺ desensitization by inhibiting KCl-induced tonic force and MLC phosphorylation without inhibiting [Ca²⁺]_i. At pCa 5 in -escin-permeabilized muscle, FSK and 8b-cGMP accelerated the relaxation rate when tissues were returned to pCa 9, suggesting that both agents can elevate MLCP activity. However, a component of the Ca²⁺ desensitization attributed to PKG activation in intact tissues appeared to involve a MLC phosphorylation-independent component. Inhibition of KCl-induced tonic force by the ROK inhibitor, Y-27632, and by PE-pretreatment, were synergistically potentiated by 8b-cGMP, but not by FSK. FSK and PE-pretreatment, but not 8b-cGMP, inhibited the KCl-induced increase in site-specific MYPT1 phosphorylation at Thr-853. These data support the hypothesis that PKA and PE-pretreatment converge on a common Ca²⁺ desensitization pathway, but that PKG can act by a mechanism different from that activated by PKA and PE-pretreatment.