The zeta potential of washed Tice substrain BCG organisms was measured over a range of ionic strengths from I = 0.005 to 0.1 M. No change in the isoelectric point of 3.4-3.7 was evident. Proteolytic enzymes (trypsin/chymotrypsin, pepsin, papain and pronase) and fluorodinitrobenzene abolished the cationic charge, suggesting that this is substantially due to amino groups associated with protein. Neither hot HCI nor cold trichloroacetic acid affected the charge, indicating that ionic groups are not associated with extractable polysaccharides. Methanolysis, treatment with HF and carbodiimide, and cationic detergent (cetyltrimethylammonium bromide) binding indicated that the negative charge was provided by carboxylic acids, phosphoesters and strong acidic groups, possibly sulphates. Standardless quantitative X-ray microanalysis revealed the presence of phosphorus and sulphur on the surface of actively growing BCG colonies.