This study describes an alpha-lactalbumin folding variant from human milk with bactericidal activity against antibiotic resistant and susceptible strains of Streptococcus pneumoniae. The active complex was purified from casein by a combination of anion exchange and gel chromatography. Unlike other casein components the active complex was retained on the ion exchanger and eluted only with high salt. The eluted fraction showed N-terminal and mass spectrometric identity with human milk a-lactalbumin, but monomeric, native a-lactalbumin had no bactericidal effect. Native a-lactalbumin could be converted to the active bactericidal form by ion-exchange chromatography under conditions used to isolate the active molecule from human milk whey. Analysis of the antibacterial spectrum showed selectivity for streptococci; Gram negative and other Gram positive bacteria were resistant.