Group A streptococci were grown in the presence of [2-3H]glycerol. Concentrated suspensions of labeled organisms were incubated with and without penicillin. [3H]Glycerol-labeled material accumulated in supernates in increasing amounts with increasing concentrations of penicillin, ranging from 0-50 U[units]/ml. Excretion of labeled material occurred in the absence of nucleic acid synthesis or bacteriolysis indicating that the phenomenon is independent of cell multiplication or decay. The accumulation of label was paralleled by an accumulation of erythrocyte-sensitizing material measured by passive hemagglutination tests for lipoteichoic acid antigen, indicating that a portion of the labeled material possessed the properties of lipoteichoic acid. Culture supernates were fractionated by column chromatography, and materials obtained were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide, TLC and paper chromatography. The ability of the same materials to bind to human erythrocytes and epithelial cells was tested. The culture supernate contained lipoteichoic acid, deacylated lipoteichoic acid, glycerol phosphate and free glycerol. Penicillin caused an increase in amounts of each of the excreted materials. Streptococci that were stimulated with penicillin to lose their lipoteichoic acid (previously shown to mediate adherence of group A streptococci) lost their ability to adhere to buccal mucosal cells, suggesting that penicillin may influence bacterial ecology by mechanisms other than killing sensitive organisms.