Aquaporin-1 (AQP1) is a membrane channel that allows rapid water movement driven by a transmembrane osmotic gradient. It was claimed to have a secondary function as a cyclic nucleotide-gated ion channel. However, upon reconstitution into planar bilayers, the ion channel exhibited a 10-fold lower single channel conductance than in Xenopus oocytes and a 100-fold lower open probability (<10(-6)) of doubtful physiological significance (Saparov, S. M., Kozono, D., Rothe, U., Agre, P., and Pohl, P. (2001) J. Biol. Chem. 276, 31515-31520). Investigating AQP1 expressed in human embryonic kidney cells, we now have shown that the discrepancy is not due to alterations of AQP1 properties upon reconstitution into bilayers but rather to regulatory processes of the oocyte expression system that may have been misinterpreted as AQP1 ion channel activity. As confirmed by laser scanning reflection microscopy, from 0.8 to 1.4 x 10(6) AQP1 copies/cell contributed to osmotic cell swelling. The proper plasma membrane localization was confirmed by observing the fluorescence of the N-terminal yellow fluorescent protein tag. Whole-cell patch clamp experiments of wild type or tagged AQP1-expressing cells revealed that neither cGMP nor cAMP mediated ion channel activity. The lack of significant CNG ion channel activity rules out a secondary role of AQP1 water channels in cellular signal transduction.