Phenylarsine oxide (PAO), a sulfhydryl modifying reagent and a widely used inhibitor for tyrosine phosphatases and endocytosis, was tested on the level of phosphorylation in human nonpigmented ciliary epithelial ocular (HNPE) cells. Pretreatment with (PAO, 10 μM) for 30 min followed by incubation with 32P(i) to stimulate endogenous phosphorylation surprisingly resulted in a total reduction in 32P(i) labeled proteins. PAO (10-50 μM) dose-dependently inhibited both sodium-dependent and -independent phosphate uptake in cells. p-Hydroxymercuribenzoate (pHMB, 10 μM), another sulfhydryl modifying reagent failed to mimic PAO effects. However, metabolic inhibitors (iodoacetamide (0.1 mM) and 2,4- initrophenol (DNP, 0.5 mM) also mimicked PAO effects, suggesting that the inhibition of ATP production may be responsible for attenuation of both phosphate uptake mechanisms. However, sodium- dependent phosphate uptake in isolated plasma membrane vesicles pretreated with PAO was also significantly lower than control vesicles treated with dimethlysulfoxide (DMSO), suggesting that PAO may be directly targeting a component of the sodium-dependent cotransporter. It is suggested that PAO is a novel inhibitor of phosphate uptake in HNPE cells that acts indirectly by inhibiting ATP production and directly by inhibiting the Na-dependent cotransporter.