Analysis of restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA (ribotyping) was used to document four cases of Streptococcus agalactiae mother-to-infant transmission potentially associated with ingestion of infected mother's milk. Twenty strains were analyzed. Ten strains were mother-baby pairs, five from the milk of five mothers, four from their neonates with late-onset infection, and one from a colonized neonate. All mothers had early postpartum mastitis. Ten unrelated strains were studied for comparison. In each case, the two strains of each mother-baby pair produced identical RFLP patterns of total DNA. The 10 unrelated strains generated 10 different patterns, one of which, though, was observed in one of the mother-baby pairs. Ribotyping was less discriminative than total DNA RFLP analysis (6 different patterns vs. 13). These data extend the evidence for breast milk transmission in S. agalactiae late-onset neonatal infection.