ABSTRACT.Purpose: Up to one-third of patients with intra-ocular silicone oil (SO) tamponade for complex macula-on retinal detachment may experience an unexplained visual loss during or after SO tamponade. Although the underlying mechanism is unknown, previous studies suggested that accumulation of retinal potassium could be involved. Hence, this study tested the hypothesis that intraocular potassium levels are elevated during SO tamponade. Methods: A prospective cohort study was carried out from 13 October 2013 through 5 March 2015. Potassium, sodium, magnesium, chloride, calcium, lactate dehydrogenase (LDH) and glucose levels were measured in retro-oil fluid and paired serum from 16 patients undergoing oil removal, including two patients with SO-related visual loss (SORVL). Vitreous humour and paired serum from 27 patients with macular hole (n = 19) or floaters (n = 8) served as controls. Results: Median potassium levels in retro-oil fluid and vitreous humour were similar. Magnesium and chloride levels were lower in retro-oil fluid compared with vitreous humour (p < 0.01) and LDH levels were elevated in retro-oil fluid (p < 0.0001). One of the two patients with SORVL revealed abnormal high potassium and magnesium levels. The other patient had normal levels. Conclusion: Potassium levels are not increased in retro-oil fluid during SO tamponade, making the 'potassium accumulation' hypothesis unlikely. The disturbance in magnesium concentration during SO tamponade warrants further investigation.