Chick lens fiber cell gap junctions were isolated to homogeneity by the urea-deoxycholate method, characterized ultrastructurally and biochemically, and their lipid composition determined by quantitative thin layer chromatography (TLC). The junctions were estimated to comprise about 52% of the lens fiber plasma membrane. Unlike the junctions of other organs, the lens gap junctions were found to contain sphingomyelin. The cholesterol/phospholipid molar ratio was 2.1 for total fiber membranes but 3.1 for the fiber gap junctions. The levels of major phospholipids in decreasing order were SPH, PC, PE, PI for fiber junctions and PE, SPH, PC, PI for total fiber membranes. The gap junctions were found to contain about 57% of the total fiber cholesterol and 53% of the total fiber sphingomyelin. The high cholesterol and sphingomyelin content suggests that lens fiber gap junctions constitute highly rigid membrane regions conferring significant constraints to the movement of their intramembrane particles (connexons) in the plane of the membrane. The findings help to explain the resistance to the crystallization of their connexons, observed so far only in lens gap junctions.