The distribution of fatty acids, proteins, polysaccharides and antigens in subcellular fractions of Mycobacterium avium is described. Significant qualitative differences in the chemical composition of the various fractions have been used to further characterize the tripartite structure of the cell wall. In the outer dense layer (POL), in addition to previously described complex amphiphatic lipids, new oligosaccharides (lipooligosaccharides?) and a major glycoprotein were located; and it was found that tuberculostearic acid (TSA) esterified the phospholipids of this outerlayer. Judging from the data, it was proposed that the phospholipids formed a basic matrix monolayer in which other compounds of the POL intercalated. It was suggested that in an aqueous environment the hydrophobic ends of the phospholipids oriented to face the mycolic acid residues of the cell wall skeletons (or CWS) to form the 12 nm thick electron transparent layer. The purified CWS contained alpha-, keto-, and dicarboxylic mycolic acids; alanine, glutamic acid and diaminopimelic acid; and arabinose and galactose. Two additional nonidentified amino acids and an unidentified sugar were found in the CWS. Also, in the CWS the fatty acids: palmitic acid (21.8%), oleic acid (4.3%), stearic acid (9.2%) and TSA (4.3%), were detected. The main fatty acids detected in the cytoplasmic membrane (CM) were palmitic (20%), oleic (14.5%) and stearic (8.6%) acids. Mycolic acids and TSA were absent in the CM phospholipids. The major proteins of the CM (86, 40, and 26 Kd proteins) were distinct from the major proteins detected in the cytosol (CYT) fraction (43, 36, and 19 Kd proteins). A 58 Kd protein was present in both the CM and the CYT. The CYT and CM antigens were found absent in surface antigens extracted using sodium dodecyl sulphate (SDS).