The effects of putative water channel blockers were tested on AQP1-expressing Xenopus laevis oocytes by a fast optical method with a time resolution of 1 s and a volume resolution of 20 pl. The oocytes were exposed to external hyposmolarity and the osmotic water permeability (Lp) derived from the initial 10 s of volume change. For longer durations, the effective osmotic gradient across the membrane was reduced significantly because of dilution of the intracellular contents and of ion transport across the membrane. The latter was monitored by voltage clamp of the oocytes. In contrast to previous reports based on slower and less sensitive assays, we found no effects of tetraethylammonium ions (TEA+) and acetazolamide on Lp. We have no single explanation for this, but several factors are considered: (a) If the osmotic gradient is assumed to be constant for periods longer than 10 s, the Lp will be underestimated. (b) Hyposmotic gradients implemented by dilution with water will entail changes in the ionic strength as well; this may enhance loss of salt from the oocyte. (c) By voltage clamping the AQP1-expressing oocytes during hyposmotic challenges, we found that TEA+-treated oocytes were more electrically leaky than untreated ones. This may obscure comparisons between the Lp of treated and untreated oocytes. (d) The nature of the ion transport mechanisms in the plasma membrane depends on how oocytes have been prepared for experiments and on their viability as indicated by the membrane potential. These parameters may vary between laboratories.