The morphologic features of third-passage human trabecular cells were examined in multiple experiments by light and electron microscopy. Evaluation of the cellular growth pattern, fibronectin immunofluorescence, and specific ultrastructural features have helped to establish the identity of the trabecular cell type. Comparison of the cultured trabecular cells with cultured keratocytes and scleral fibrocytes grown under identical conditions emphasized the specialized morphologic features of trabecular cells in vitro. The characteristics of trabecular cells in vitro closely resemble those described for trabecular cells in vivo, including the presence of similar cell surface features and cell junctions, cytoplasmic organelle composition, and nuclear chromatin pattern. Since many of the differentiated features of the trabecular cells are retained after passage in culture, it is possible that study of these cells will provide a means to understand the biochemical and morphologic properties of this important cell type.