As reported by others (e.g. Rembold et al., 2000. J Appl Physiol 91, 1460-1466), we found in gastric fundus by 2D-PAGE analysis that cyclic nucleotide-mediated relaxation is only transiently associated with dephosphorylation of MLC₂₀. Electrical field stimulation of ET-1-precontracted fundus strips led to relaxation which amounted to 4% after 2, 31% after 5 and 84% after 30 sec of EFS compared to ET-1 induced contraction. In contrast, MLC₂₀ monophosphorylation initially declined from 35% to 18% after 2 sec of EFS but thereafter returned to its initial value under ET- 1-contraction (30 sec: 33%). MALDI-TOF-MS and LC- MS/MS analyses allocated the monophosphorylation to the first three amino acids of peptide 17-34 including Ser19 and Thr18 but could not discriminate between these two sites. Immunodetection with phosphospecific antibodies showed that Ser19 phosphorylation decreases during EFS and remains low, while the increase in phosphorylation, seen with 2D-PAGE, is due to Thr18 phosphorylation. Since fura-2 measurements revealed a decline of intracellular [Ca²⁺] during relaxation, Thr18 appears to be rephosphorylated in a Ca²⁺-independent manner. In summary, we showed that (i) Ser19 dephosphorylation correlates to relaxation and (ii) MLC₂₀ can be monophosphorylated not only at Ser19 but also at Thr18.