Human breast milk samples were collected from lactating mothers, and aliquots were maintained at room temperature, frozen, and pasteurized. Samples were inoculated with 10 to 50 colony-forming units per milliliter (cfu/ml) of Escherichia coli or group B streptococcus, and incubated at 37 C. Quantitative growth was measured at eight and 24 hours. No inhibitory activity was demonstrated by control broth, commercial formula, and pasteurized breast milk, which had a rapid logarithmic growth to a maximum of 10(8) to 10(9) cfu/ml at 24 hours. Compared with these controls, fresh breast milk, fresh frozen breast milk, and breast milk frozen for 21 days demonstrated a significant inhibition of bacteria growth. A trend toward gradual loss of inhibiting activity was noted with prolonged freezing of breast milk. Although freezing may quantitativly decrease the amount of some breast milk host-defense factors, it cannot be assumed that comparable functional reductions will necessarily result.