Intraocular pressure (IOP) is regulated by the resistance to outflow of the eye's aqueous humor. Elevated resistance raises IOP and can cause glaucoma. Despite the importance of outflow resistance, its site and regulation are unclear. The small size, complex geometry and relative inaccessibility of the outflow pathway have limited study to whole-animal, whole-eye or anterior-segment preparations, or isolated cells. We now report measuring elemental contents of the heterogeneous cell types within the intact human trabecular outflow pathway using electron-probe X-ray microanalysis. Baseline contents of Na⁺, K⁺, Cl^- and P, and volume (monitored as Na+K contents) were comparable to those of epithelial cells previously studied. Elemental contents and volume were altered by ouabain to block Na⁺,K⁺-activated ATPase and by hypotonicity to trigger a regulatory volume decrease (RVD). Previous results with isolated trabecular meshwork (TM) cells had disagreed whether TM cells express an RVD. In the intact tissue, we found that all cells, including TM cells, displayed a regulatory solute release consistent with an RVD. Selective agonists of A₁ and A₂ adenosine receptors (ARs), which exert opposite effects on IOP, produced similar effects on juxtacanalicular (JCT) cells, previously inaccessible to functional study, but not on Schlemm's canal cells that adjoin the JCT. The results obtained with hypotonicity and AR agonists indicate the potential of this approach to dissect physiologic mechanisms in an area that is extremely difficult to study functionally, and demonstrate the utility of electron microprobe analysis in studying the cellular physiology of the human trabecular outflow pathway in situ.