In this report the cell wall of Mycobacterium avium is shown as a triple-layered structure where the outermost layer was stained by the ruthenium red staining for polysaccharides. The outermost layer hindered the diffusion of chemotherapeutic agents across the wall thus causing multiple drug-resistance by exclusion. The concerted electron microscopy and chemical analysis of chloroform-methanol and Triton X-100 extracts indicated that the outer layer was made of diverse amphiphil glycolipids (mycosides C, glycolipids, peptidolipids, phospholipids) that formed a matrix in which proteins were embedded. The examination of a spontaneous rough mutant indicated that mutations blocking the synthesis of one or several of the amphiphils must leave unsubstituted mycolic acid residues, thus causing surface hydrophobicity and roughness. Judging from our data, a model describing the overall cell envelope of M. avium was proposed. From the comparative analysis of M. avium, its spontaneous rough mutant, and its spheroplasts, some of the functions of the outermost layer were disclosed.
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