The nutritionally variant streptococci (NVS) are a fastidious group of bacteria first recognized in the early 1960s in the blood cultures of patients with subacute bacterial endocarditis. Since that time, the NVS have been implicated in 5 to 6% of all cases of human bacterial endocarditis. The NVS possess membrane-associated amphipathic molecules different from those described for other streptococci. Unitl recently, chemical characterization of these new amphipathic polymers was hampered by unsuccessful attempts at isolating large quantities of these molecules in a form free from other bacterial components. Presently, stationary-phase-culture supernatants provide an optimum source of crude material for amphiphile purification procedures. Hydrophobic-interaction chromatography in conjunction with immunoaffinity chromatography yields an NVS serotype I amphiphile preparation free of contaminants, as determined by immunoelectrophoretic and chemical analyses. Tandem crossed immunoelectrophoresis of the purified extracellular NVS amphiphile demonstrated that it is immunologically similar to the intracellular amphiphile. Finally, this amphiphile serves as the NVS serotype I antigen.