To determine seropositivity for human T-lymphotropic virus type I (HTLV-I), we attempted to improve the detection system that uses antibody to HTLV-I Env in Western immunoblotting (WB) by adding an envelope glycoprotein (gp46) purified from the culture fluid of HTLV-I-producing cells by immunoaffinity chromatography and gel chromatography. In this WB, 177 of 179 serum samples showing seropositivity in an indirect immunofluorescence assay showed positive reactions to the gp46 envelope antigen as well as to p19, p24, and p53 Gag antigens. The remaining two samples showed negative reactions to p24. False-positive results were not found for 533 indirect immunofluorescence assay-negative serum samples, although one band to p19 or p24 was observed in 46 of the 533 samples. These 46 samples did not react to p53 and gp46, suggesting that these samples belonged to the indeterminate group in accordance with the criteria proposed by the World Health Organization. Therefore, this improved WB can be used for the confirmation of seropositivity.