Trabecular meshwork cell cultures have been grown from excised calf aqueous outflow pathway tissue using two different methods. In one method, tissue explants were placed directly in culture dishes and primary cultures were established by those cells migrating from the tissue. In the second method, a cell suspension was generated by incubating trabecular tissue strips with 0.1% pronase, and the washed cells were used to seed primary cultures. The cell cultures generated by each method appeared to progress through morphological and biochemical changes characteristic of the cell type, although at different respective rates. Comparisons of the cell morphologies, biosynthetic activities, and growth characteristics of the respective subcultures suggested that similar cell populations were obtained by each method. These studies further suggested that sufficient quantities of calf trabecular cells could be grown in culture to permit biochemical and pharmacological studies of trabecular-cell metabolism.