Background/Aims: To evaluate the retinal toxicity of Brilliant Blue G (BBG) following intravitreal injection in rat eyes and examine the biocompatibility and the staining properties in humans.Methods: BBG was injected into the 11 rat eyes to evaluate toxic effects with balanced salt solution (BSS) serving as control. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts and by light microscopy 7 days later. In addition, BBG was applied during vitrectomy for macular hole (MH) (n = 15) or epiretinal membranes (ERM) (n = 3) in a prospective, non-comparative consecutive series of patients. Before and after surgery, all patients underwent a complete clinical examination including measurement of best corrected visual acuity (VA) and intraocular pressure, perimetry, fundus photography and optical coherence tomography. Patients were seen 1 day before surgery and then in approximately four weeks intervals.Results: No significant reduction in RGC numbers and no morphological alterations were noted. A sufficient staining of the internal limiting membrane (ILM) was seen in patients with MH, while the staining pattern in ERM cases was patchy, indicating that parts of the ILM were peeled off along with the ERM in a variable extent. All MHs could be closed successfully. VA improved in 10 eyes (56%; 8/15 MH patients, 2/3 ERM patients), was unchanged in four eyes (22%; all MH patients) and was reduced in four eyes (22%; 3/15 MH, 1/3 ERM). No toxic effects attributable to the dye were noted during patient follow-up. The ultrastructure of tissue harvested during surgery was unremarkable.Conclusion: Brilliant Blue provides a sufficient and selective staining of the ILM. No retinal toxicity or adverse effects related to the dye were observed in animal and human studies. The long-term safety of this novel dye will have to be evaluated in larger patient series and a longer follow-up.