In this study, we investigated the signalling events which inactivate the smooth muscle contractile machinery of endothelin-1 (ET-1) precontracted GF upon electrical field stimulation (EFS) activating NANC neurons. EFS induced a rapid fall in tension (t_1/2: 7.8 sec) which was preceded by a decrease in intracellular [Ca²⁺], dephosphorylation of myosin light chains (MLC₂₀) at Ser19 and of the regulatory subunit (MYPT1) of MLC phosphatase at the inhibitory Rho kinase (ROK) sites Thr696 and Thr853. The latter event cannot be ascribed to inactivation of Rho because the amount of GTP- loaded Rho remained high for > 10 sec. In addition, EFS led to phosphorylation of the PKG/PKA substrates telokin and MYPT1 at Ser695 und interestingly to monophosphorylation of MLC₂₀ at Thr18. Phosphorylation of these substrates progressed slower than the time course of relaxation. In conclusion, NANC neuron induced relaxation involves a simultaneous inactivation of MLCK and rapid activation of MLCP mediated by desphosphorylation of the ROK sites of MYPT1. It is tempting to speculate that dephosphorylation of MYPT1 is due to activation of a MYPT1 phosphatase.