Mycoplasma fermentans is thought to be a pathogen of rheumatoid arthritis or cofactor of AIDS. A novel phosphocholine-containing glycoglycerophospholipid named GGPL-I was isolated from a M. fermentans-infected human helper T-cell culture. It was revealed that GGPL-I is a lipid component of the M. fermentans and a major immunological determinant. The GGPL-I was purified by DEAE-Sephadex column chromatography and repeated Iatrobeads column chromatography. The purified glycophospholipid was subjected to structural characterization by thin-layer chromatography, Fourier-transform infrared spectrometry, liquid secondary ion mass spectrometry, and nuclear magnetic resonance spectroscopy. Its structure was determined to be as follows: 6'-O-phosphocholine-alpha-glucopyranosyl-(1'-3)-1,2-diacyl-sn-glycerol. This glycoglycerophospholipid is unique in containing phosphocholine, which is attached to C-6 of glucose. The stereospecific numbering (sn) of naturally occurring GGPL-I was determined through comparison with chemically synthesized compounds.