We report for the first time that cultured nonpigmented human ciliary epithelial (NPE) cell layers transport fluid. Cells were grown to confluence on permeable membrane inserts, and fluid transport across the resulting cell layers was determined by volume clamp at 37°C. These cell layers translocated fluid from the apical to the basal side at a steady rate of 3.6 µl · h¹ · cm² (n = 4) for 8 h. This fluid movement was independent of hydrostatic pressure and was completely inhibited by 1 mM ouabain, suggesting it arose from fluid transport. Mercuric chloride, a nonspecific but potent blocker of Hg²⁺-sensitive aquaporins, and aquaporin-1 antisense oligonucleotides both partially inhibited fluid transport across the cell layers, which suggests that water channels have a role in NPE cell homeostasis. In addition, these results suggest that of the two ciliary epithelial layers in tandem, the NPE layer by itself can transport fluid. This cultured layer, therefore, constitutes an interesting model that may be useful for physiological and pharmacological characterization of ciliary epithelial fluid secretion.