A method which is capable of accurately determining low amounts of protein (i.e., 2-24 rg) in the presence of very high levels of lipid (i.e., 20-40 mg) has been developed. The procedure was developed from the original amido black 10B protein assay of Schaffner and Weissmann (W. Schaffner and C. Weissmann, 1973, Anal. Biochem. 56,502-S 14) and incorporates several critical modifications that enable the assay to be performed with lipid-containing samples without interference. The modifications include a substantial increase in the assay volume (thereby decreasing the final lipid concentration) as well as the sodium dodecyl sulfate and trichloroacetic acid concentrations. Under these conditions, a linear standard curve is obtained with 2-24 pg of bovine serum albumin in both the absence and the presence of lipid (20 mg). Moreover, the assay is unaffected by as much as 40 mg of lipid in the original sample. Linearity as well as noninterference by lipid (20 mg) is also demonstrated with a sample of mitochondriat protein (i.e., a mixture of hydrophilic and hydrophobic proteins). Additionally, we show that in the presence of protein (20 pg) and lipid (20 mg), high concentrations of various buffers, salts, and nonionic detergents do not interfere with the assay. Finally, the enhanced ability of this new method to tolerate high lipid levels without interference relative to several existing protein estimation methods is demonstrated. This procedure should prove widely useful for measuring protein in reconstituted systems involving proteoliposomes. Such studies often require the incorporation of small amounts of protein into liposomes in the presence of detergent and under conditions where a very high lipid/ protein ratio exists. To our knowledge, this is the first demonstration that a spectrophotometric assay is capable of accurately measuring low amounts of protein in the presence of such high concentrations of lipid. 0 1985 Academic PES, h.