The frequency and extent of immunostaining for actin, fibronectin (FN), and glial fibrillary acidic protein (GFAP) were determined in 37 preretinal membranes (PRMs) obtained at vitrectomy from 35 patients with proliferative diabetic retinopathy (PDR) (n = 16), proliferative vitreoretinopathy (PVR) (n = 18), or idiopathic macular pucker (MP) (n = 3). All three proteins were detected in the vast majority of specimens (actin, 86%; FN, 95%; GFAP, 96%), although the extent of staining varied for each. Actin-FN co-localization was observed in all diagnostic groups on comparison of adjacent sections and in double-labeled sections. The extent of actin staining did not correlate with clinical grading of PRM contraction. In PDR membranes, FN staining was low overall, but proportional to the vascular content of the PRM. Fibronectin staining of PVR membranes was greater, and extensive even in avascular specimens. In MP membranes, most cells were GFAP-positive, whereas in PDR and PVR specimens, GFAP staining was variable. The lack of correlation of clinical contractility and membrane composition, as studied in this article by immunostaining, indicates that other factors must play significant roles in determining membrane behavior.