Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a K-d of 1.2 +/- 0.2 nM and B-max of 110 +/- 5 pmol/mg for [H-3]nitrobenzylmercaptopurine ribonucleoside ([H-3]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a K-d of 4.3 +/- 0.7 nM. The yield of hENT1 from Sf9 cells was similar to 0.5 mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. (C) 2015 Elsevier Inc. All rights reserved.