Despite widespread evidence for T cell autoreactivity to MHC-peptide complexes and certain CD1 proteins, autoreactivity to human CD1b proteins has not been documented. Using CD1b dextramers loaded with polar lipids from Staphylococcus aureus and Salmonella typhimurium, we isolated CD1b-reactive T cell lines from human blood donors. Surprisingly, the CD1b-reactive T cell clones showed autoreactivity to human CD1b proteins expressed on mammalian cells in the absence of bacteria. Such autoreactive responses suggested the existence of lipid autoantigens for the human CD1b system. To identify lipid autoantigens, we loaded CD1b dextramers with the major biochemical classes of mammalian phospholipids. Similar to recent results in the CD1a system, T cells showed cross-reactivity to several structurally related lipid subclasses. However, T cell lines showed preferential activation and T cell receptor binding to CD1b loaded with phosphatidylglycerol (PG), while they also recognized phosphatidic acid (PA). Using T cell receptor transfer and purified and synthetic mammalian and bacterial PGs, we established that the response is TCR-mediated and shows cross-reactivity to self and bacterial PGs that differ in fatty acid composition. In mammalian cells, PG is an intermediate in cardiolipin metabolism and is largely sequestered in mitochondria. T cell autoreactivity to CD1b proteins with or without lysosomal targeting sequences suggested organelle-specific rather than ubiquitous capture of self lipids. Thus, human ab T cells can recognize CD1b, which presents phospholipid antigens that are rare or sequestered in mammalian cells, but are abundant components of bacteria.