A rapid, sensitive spectrophotometric method for the analysis of sulfatides is described. The assay method is based upon the formation of a colored complex between the cationic dye azure A and (anionic) sulfolipids; the complex is extractable by a solution of chloroform-methanol 1:1. With the exception of some of the phospholipids, the reaction is specific for the sulfolipids. Cardiolipin was the most reactive of the non-sulfolipid compounds that were tested. Except for sphingosine and the mono- and dihexose derivatives of sphingosine, a wide variety of lipids did not interfere with the formation of color by standard sulfatides. Sulfur-containing amino acids, sugar sulfates, and sulfated polymers neither produced color nor interfered in its formation by sulfatide. Chloride and nitrate ions produced relatively little color. The method is much more sensitive (lower limit, about 0.002 micromole) than most methods currently employed for the analysis of sulfatides and has a high degree of precision. It is applicable to the analysis of sulfolipids in tissue extracts and gives values similar to those obtained by previously published procedures. The reliability of the method is increased when it is applied to partially purified lipid solutions. Sulfatides added to extracts of rabbit brain tissue were quantitatively accounted for by the assay. In addition, the method can be applied to samples obtained directly from thin-layer plates.