The location of the pX gene products in human T-cell leukemia virus type 1-producing cells, MT-2 and HUT 102, was studied by immunoelectron microscopy using the direct and indirect peroxidase-labeled antibody methods. Fab'-peroxidase conjugates were prepared for the direct method with a maleimide compound from antisera to the carboxy-terminal region of the pX gene products. Positive immunostaining in MT-2 cells was detected in the endoplasmic reticulum, the outer and inner leaflets of the nuclear membrane, and inside their cisternae, but not in the plasma membrane and viral particles. Staining in the nucleus was faint. On the other hand, positive immunostaining in HUT 102 cells was detected diffusely in the euchromatin regions of the nucleus but not in the nucleoli, nuclear envelope, and cellular membrane systems. The location of the positive immunostaining in the HUT 102 nuclei was reconfirmed by the reaction in isolated nuclei. On the basis of both the immunoelectron microscopic and immunoblotting analyses of the pX gene products, it is suggested that the Mr 40,000 to 42,000 protein (p40x) is localized mainly in the euchromatin regions of the nuclei of human T-cell leukemia virus type 1-producing cells, and the Mr 68,000 protein (p68x) is localized mainly in the nuclear envelope and the endoplasmic reticulum of MT-2 cells. p68x detected in MT-2 cells with the anti-p40x serum was deduced to be a protein consisting of p40x and a part of env gene products and to share epitopes in common with p40x.