The purpose of this study was to compare steroid hormone concentration levels in the vitreous and serum of vitreoretinal disease patients to elucidate the possibility of neurosteroid production in the retina. Serum and vitreous samples were collected from vitrectomy patients, and estradiol (E2) and testosterone (T) concentrations were measured using electro-chemiluminescence immunoassay. We measured E2 in epiretinal membrane (ERM, n = 14), macular hole (MH, n = 18), proliferative diabetic retinopathy (PDR, n = 20), and retinal detachment (RD, n = 19) cases, and T in ERM (n = 14), MH (n = 17), PDR (n = 13), and RD (n = 17) cases. No statistically significant age differences existed among the groups. Mean respective E2 concentrations (pg/ml) in the male/female vitreous were ERM: 6.67 +/- 4.04/18.82 +/- 7.10, MH: 10.3 +/- 7.02/17.00 +/- 4.8, PDR: 4.2 +/- 3.05/15.83 +/- 3.46, and RD: 10.00 +/- 4.58/16.06 +/- 4.57, while those in serum were ERM: 31.67 +/- 5.51/5.82 +/- 1.08, MH: 21.00 +/- 8.89/7.53 +/- 3.2, PDR: 29.20 +/- 7.07/12.75 +/- 10.62, and RD: 24.33 +/- 6.51/7.5 +/- 4.42. E2 concentrations were significantly higher (P<0.001) in the male serum than vitreous, yet significantly higher in the female vitreous than serum. Mean respective T concentrations (ng/ml) in the male/female vitreous were ERM: 0.15 +/- 0.03/0.15 +/- 0.01, MH: 0.15 +/- 0.01/0.15 +/- 0.01, PDR: 0.15 +/- 0.03/0.16 +/- 0.12, and RD: 0.14 +/- 0.01/0.17 +/- 0.08, while those in serum were ERM: 4.54 +/- 1.46/0.16 +/- 0.01, MH: 8.04 +/- 2.29/0.16 +/- 0.10, PDR: 5.14 +/- 1.54/0.22 +/- 0.11, and RD: 3.24 +/- 0.75/0.17 +/- 0.10. T concentrations were high in the male serum, yet extremely low in the male and female vitreous and female serum. High concentrations of E2 were found in the vitreous, and women, in particular, exhibited significantly higher concentrations in the vitreous than in the serum. This finding suggests the possibility that in vitreoretinal disease cases, the synthesis of E2 is increased locally only in female eyes.