1 The mechanisms of action on smooth muscle of the iron-sulphur cluster nitrosyl compound, heptanitrosyl-tri-gL3-thioxotetraferrate (1-), (RBS), a photosensitive nitric oxide donor, have been investigated in the guinea-pig taenia caeci (coli) in vitro. 2 After exposure to RBS (50 I1M) for 30 min, and subsequent washout, a sustained contraction was recorded in the absence of light to either the agonist carbachol (50 pLM) or a depolarizing concentration of KCI (23.5 mM). Photon irradiation (>400 nm) caused a prompt relaxation of precontracted RBStreated muscle, the magnitude of which depended upon the intensity (1.1 x 103 to 1.1 x i0s lux), duration (30 s to 20 min) and wavelength (400 to 800 nm), of the incfdent illumination.3 Repeated periods of illumination at 1.1 x 104 lux produced a reversible relaxation of both carbachol and KCl-evoked tone in muscle pretreated with RBS (50 psM). These photorelaxations were reproducible at 10 min intervals for several hours with a maximal relaxation amounting to 80 to 90% that of the tone produced by carbachol (50 gM). 4 The nitric oxide synthase inhibitor, N0-nitro-L-arginine (60 tM), caused no inhibition of the photoninduced relaxation of RBS-treated muscle. In contrast, N-methylhydroxylamine (2 mM), L-cysteine (10 mM), DL-dithiothreitol (2 mM), methylene blue (30 ptM), and haemoglobin (20 SiM), all reversibly but significantly inhibited (P<0.001) the photorelaxation response. However, neither the aminothiol Nacetyl-L-cysteine (10 mM) nor the tripeptide glutathione (10 mM) blocked the RBS-induced photorelaxation. 5 The photolytic cleavage of RBS depended on the intensity and duration of illumination; it was accompanied by a corresponding decrease in absorbance and by the liberation of NO as measured by the Griess diazo reaction with sulphanilic acid. L-Cysteine (10 mM) prevented the decrease in absorbance and the photolytic liberation of NO. 6 It is concluded that (i) sequestered or bound RBS, when photon-activated, liberates NO by a process which can be controlled by the wavelength, intensity and duration of the incident light, (ii) the photon-released NO rapidly relaxes the smooth muscle cells of the taenia coli primarily via cyclic GMP-dependent pathways which can be blocked by use of appropriate inhibitors, and (iii) the RBS-induced photorelaxation effect does not involve the activation of NO synthase. RBS is therefore a valuable photosensitive NO donor for establishing the functional and pharmacological significance of NO.