The lanthanide ions La(3+) and Gd(3+) block Ca(2+)-permeable cation channels and have been used as important tools to characterize channels of the transient receptor potential (TRP) family. However, widely different concentrations of La(3+) and Gd(3+) have reportedly been required for block of TRP3 channels in various expression systems. The present study provides a possible explanation for this discrepancy. After overexpression of TRP3 in Chinese hamster ovary cells, whole-cell currents through TRP3 were reversibly inhibited by La(3+) with an EC(50) of 4 microm. For comparison, the organic blocker SKF96365 required an EC(50) of 8 microm. Gd(3+) blocked with an EC(50) of 0.1 microm, but this block was slow in onset and was not reversible after wash-out. When the two lanthanides were added to the cytosolic side of inside-out patches, block was achieved with considerably lower concentrations (EC(50) for La(3+), 0.02 microm; EC(50) for Gd(3+), 0.02 microm). Uptake of La(3+) into the cytosol of Chinese hamster ovary cells was demonstrated with intracellular fura-2. We conclude that lanthanides block TRP3 more potently from the cytosolic than from the extracellular side of the plasma membrane and that uptake of lanthanides will largely affect the apparent EC(50) values after extracellular application.