To investigate if human anterior lens capsule is a suitable substrate for the culture of primary human trabecular meshwork (HTM) cells. Trabecular meshwork cells derived from four human donors were seeded on anterior lens capsules that were prepared from the lenses of donor eyes. Cell morphology and viability were examined at 1, 3, 5 and 7 days. Cell viability was measured based on a two-colour fluorescence assay (membrane-impermeable propidium iodide and membrane permeable Hoechst 33342). Immunocytochemistry studied Zonula occludens-1 (ZO-1), vimentin, tissue transglutaminase (tTgase) and Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase). Morphology of the cultivated cells followed a typical model while their viability was > 95% in all cases. ZO-1 was found at the cell boundaries of the HTM-AC complex. Vimentin was located at the lateral membranes of the HTM cells. Na(+)/K(+)-ATPase was found at the basolateral membrane of the HTM cells. tTgase was also identified. Anterior lens capsule can be considered as a suitable alternative substrate for cultivation of HTM cells and assist the expansion of existing knowledge about glaucoma pathophysiology and therapy.