We examined the extracellular matrix formation and lectin-binding properties of cultured trabecular-meshwork cells established from cynomolgus monkey and bovine eyes. Using an avidin-biotin complex method, we found that the extracellular matrix in both the monkey and bovine cultures stained intensely with antibodies to fibronectin, type IV collagen, and laminin. These materials were especially prominent when the cultured monkey trabecular-meshwork cells aggregated in clusters, manifesting their close anatomic relationship to trabecular beams. Cell-surface and intracellular lectin-binding properties were studied using eight biotinylated lectins: concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), Dolichos biflorus agglutinin (DBA), soy bean agglutinin (SBA), Phaseolus vulgaris (PHA), wheat germ agglutinin (WGA), and Ulex europaeus agglutinin I (UEA I). Both monkey and bovine trabecular-meshwork cells showed positive cell surface and intracellular binding to Con A, WGA, and PHA. Only moderate cell-surface staining was observed with RCA, and no visible staining occurred with PNA, DBA, SBA, and UEA-1.