Although trabecular meshwork cells are presumed to play an important role in determining ocular aqueous outflow resistance, little is known about their membrane transport characteristics. As in vivo access by microelectrodes is difficult, we used cell culture techniques to facilitate membrane voltage recording from cultured bovine trabecular meshwork cells. Phase-contrast microscopy revealed the presence of epithelial-like and spindle-shaped cell types. The mean membrane voltage for epithelial cells was -49.7 +/- 0.8 mV (S.E.M., n = 143) and for spindle cells was -70.9 +/- 1.9 mV (S.E.M., n = 48). These cells possess an electrogenic Na+/K(+)-ATPase and a K+ conductance. K transference numbers (tk) for [K+] from 5 to 80 mM were 0.50 for epithelial cells and 0.71 for spindle cells. The epithelial cells lack the electrogenic Na+/HCO3-symport, thereby enabling their differentiation from corneal endothelial cells and confirming previous reports of differences between these cell types. A proportion of spindle cells demonstrated spontaneous and induced fluctuations of membrane voltage. One millimolar Ba2+ (n = 9) induced an immediate depolarization of membrane voltage, with the onset of 'overshooting' action potentials, which were dependent on extracellular Ca2+ and Na+ but were not blocked by tetrodotoxin, 10(-6) M. Spindle cells showed parallel alignment of intracellular smooth muscle specific alpha-isoactin filaments, whereas epithelial cells showed specks of non-fibrous staining. Electron microscopy revealed that epithelial cells had the characteristics of metabolically active cells, with few intermediate filaments (10-12 nm) and microfilaments (6-7 nm) and short cytoplasmic processes. Spindle cells had long cytoplasmic processes and abundant intermediate- and microfilaments. These data provide further evidence for multiple bovine trabecular cell types. The smooth muscle-like spindle cell may represent the previously proposed contractile element of the angle and its action could conceivably alter ocular outflow resistance.