The present study was undertaken to address the question whether lipid differentiation occurs in junctional domains which could imply a functional requirement for specific lipids in junctional structures. Junction enriched membranes were isolated from bovine lens fiber cells using Tris and urea treatment, and the presence of junctional structures was ascertained by electron microscopy. Enrichment in major intrinsic protein (MIP, MP26) was monitored by SDS polyacrylamide gel electrophoresis. Junctional lipids were extracted by a modified Folch procedure, to quantitatively recover cholesterol, and lipid classes were analyzed. While 99.5% of total lens protein was solubilized in the course of junction isolation, 43.9% of cell phospholipids (PL) and 64.1% of cell cholesterol (Chol) were conserved. Cholesterol was by far the predominant lipid in the junction enriched lens fiber cell membranes (833 nmol/mg protein) and was more abundant than all phospholipids combined (682 nmol/mg protein). In isolating the junctional membranes, cholesterol levels increased 144-fold, and average phospholipid levels increased 99-fold, which resulted in an increase in Chol/PL ratio from 0.84 to 1.22. Different phospholipids showed substantially different degrees of enrichment with highest enrichments seen for the phosphatidylethanolamine fraction (152-fold) and sphingomyelin (101-fold). Thus, the phospholipids of the junction enriched membranes consisted mainly of ethanolamine glycerophospholipids (37.3%) and sphingomyelin (28.6%), with lesser amounts of choline glycerophospholipids (23.5%) and phosphatidylserine (9.2%) present. Our data suggest that the MP26 junction enriched membranes of bovine lens fiber cells contain differentiated lipid domains, and that cholesterol, ethanolamine glycerophospholipids and sphingomyelin are the prevalent boundary lipids of the major intrinsic protein in these domains.