Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Envs) interact with the CD4 receptor and CCR5/CXCR4 coreceptor expressed on target cells to mediate viral entry. Infection is initiated when the HIV-1 gp120 subunit of Envs binds to host receptors. gp120 can also be expressed as a soluble protein and is routinely used for different biophysical and immunization studies. Here, we compared the transient and stable expression of monomeric HIV-1AD8 gp120 in two closely related 293 cell lines grown in suspension. We used an HIV-1AD8 gp120-expressing plasmid to generate 293 cell lines that stably expressed HIV-1AD8 gp120 under selection of puromycin. In parallel, we used polyethyleneimine (PEI) to transiently transfect FreeStyle 293 F cells with the identical HIV-1AD8 gp120-expressing plasmid. Stable 293-AD8gp120 cells grew in media complemented with a feed supplement to a density of >1E7 cells/mL and produced an average of ~150 mg of gp120/liter of the culture, which was about 50-fold higher than the yield of gp120 produced by transient transfection of FreeStyle 293 F cells. SDS-PAGE analysis of HIV-1AD8 gp120 produced by the two different methods showed similar patterns, but gp120 purified from the stable cell line exhibited higher homogeneity. Binding of anti-gp120 antibodies to gp120 that was produced by the two methods was comparable according to ELISA. Both gp120s could bind equally well to soluble CD4 and compete with infection of viruses pseudotyped with HIV-1AD8 Envs. Our findings highlight multiple advantages of producing HIV-1AD8 gp120 by stable expression for downstream applications.IMPORTANCEThere are approximately 40.8 million people living with HIV-1 (PLWH) worldwide, with an estimate of about 1.3 million new HIV-1 infections in 2024, highlighting the urgent need for an effective HIV-1 vaccine and cure strategies. Here, we describe a highly efficient method to produce soluble HIV-1 gp120, which is intensively used in viral assays and for vaccine development. The method may be helpful in research work that needs high amounts of proteins for diverse experiments.