To express and characterize the function of a plant ion channel gene in plant cells, it is necessary to establish a model system that lacks the endogenous channel activity and can be genetically transformed. Patch-clamp techniques were used to survey voltage-dependent K+ channel activities in different cell types of tobacco plants. Interestingly, mesophyll cells lacked the inward K+ current found in guard cells. A transgene containing the inward K+ channel gene KAT1 from Arabidopsis was constructed and expressed in the mesophyll cells of transgenic tobacco plants. Expression of the KAT1 gene produced a large voltage-dependent inward current across the plasma membrane of mesophyll protoplasts. The KAT1 current was carried by K+ and activated at voltages more negative than −100 mV. This K+ current had a single-channel conductance of 6-10 pS and was highly sensitive to TEA, Cs+ and Ba2+. This study represents the first example in which a plant ion channel gene is functionally expressed and studied in plant cells. Tobacco mesophyll cells will provide a useful model for functional characterization of inward K+ channel genes from higher plants.