Background: Cyclic guanosine monophosphate (cGMP) is produced in different retinal cells, including photoreceptor cells, wherein cGMP mediates photo-transduction. CGMP is degraded by phosphodiesterases (PDE). The aim was to investigate whether retinal detachment alters intraocular cGMP levels in human eyes. Methods: cGMP and PDE were determined in vitreous fluid from 50 eyes with a retinal detachment (group I) and in 20 control samples (group II) of vitreous fluid from eyes without retinal detachment. Group III consisted of subretinal fluid samples from 70 eyes with retinal detachment. Results: cGMP in vitreous fluid from eyes with retinal detachment (6.5 (SD 1.7) nM) was decreased compared to controls (67.1 (10.0) nM) (p,0.0001). In subretinal fluid, the mean level of cGMP was 2.4 (0.2) nM. No PDE could be detected in any of the intraocular fluid samples of patients nor controls. A decrease in the mean level of cGMP in subretinal fluid of eyes with retinal detachment correlated with a longer duration of detachment (r = 20.45, p = 0.007). Conclusions: Retinal detachment was found to be associated with a decrease in vitreous cGMP concentration. In subretinal fluid, a low cGMP level correlated inversely with the duration of the detachment.