Aqueous humor outflow in primate eyes can be facilitated by ciliary muscle contraction, thereby widening fluid pathways through the trabecular meshwork. Recently in the scleral spur smooth muscle (sm) alpha-actin positive myofibroblast-like cells have been described which are in contact with the elastic fiber system of both the spur and trabecular meshwork. In the vicinity of these cells nerve terminals have been described. It is speculated that contraction of scleral spur cells can facilitate aqueous humor outflow, too. To provide a tool for further physiological and pharmacological studies monolayer cell cultures of human scleral spur have been established and characterized. For this purpose, cells derived from scleral spur, outer and inner trabecular meshwork and ciliary muscle tips from 7 donor eyes (43-87 years-old respectively, obtained 3-7 h post mortem) were grown in tissue culture medium and the different monolayer cells classified by their growth characteristics, and by immunohistochemical staining for vimentin, alpha-sm-actin, desmin, and alpha B-crystallin, respectively. In addition, the presence of alpha B-crystallin mRNA and desmin mRNA was verified using the polymerase chain reaction (PCR)-method. We were able to characterize and distinguish human scleral spur cells from adjacent ciliary muscle and trabecular meshwork cells. Scleral spur cells (SPC) grew slower than ciliary muscle cells (CMC) but much faster than trabecular meshwork cells (TMC). All cells showed the same staining characteristics in vitro as they did in vivo. Scleral spur cells stained for vimentin and alpha-sm-actin, but not for desmin and alpha B-crystallin. The corresponding mRNAs of the latter two proteins could not be detected by PCR in the spur cells. Cells grew out from all donor eyes so that they actually provide a tool for further experimental studies.