ABSTRACT. The effect of breast-feeding on the development of lymphocyte responsiveness in infants has been studied. Peripheral blood mononuclear cells from 15 breast-and 15 bottle-fed infants were obtained sequentially between 6 days and 9 months of age. A number of agents were used to stimulate the cells in vitro and the resulting proliferative responses were compared between the two feeding groups. A hanging drop microculture system using serum-free medium, enabled spontaneous proliferation and proliferative responses to several stimuli ( T and B cell mitogens, allogeneic lymphocytes, and antigen) to be studied a t a range of cell concentrations and days of culture. Significant age-related differences were found between the responses of cells from the two feeding groups. Spontaneous proliferation and proliferative responses to the T cell mitogen phytohaemagglutinin and the antigen tetanus toxoid were significantly greater in the breast-fed group a t the two earliest ages studied (6 days and 6 wk). Responses to mitogens which predominantly affect B cells, such a s pokeweed mitogen and Staphylococcus aureus (Cowan), were similar in both feeding groups a t this age. In contrast, from 3 to 9 months of age, responses of cells from bottle-fed infants were significantly greater to all stimuli than responses from breast-fed infants. One possible explanation for the higher level of proliferation by cells from newborn breast-fed infants, is that these infants may absorb the cell-growth factors and lymphokines known to be present in human colostrum and milk. These factors may stimulate T cells and/or their precursors in vivo. The subsequent greater responsiveness of peripheral blood mononuclear cells from the bottle-fed group a t 3 to 9 months of age appears to be due to a higher level of antigenic stimulation in vivo in this group. This could result from the greater intake of cow milk proteins or from the absence of the passive protective effect of the antimicrobial components in human milk. Breast-fed infants have a lower incidence of infections than bottle-fed infants (1-3). This has been attributed to a passive protective role of antimicrobial compounds in human milk, such as secretory IgA, acting locally within the gut of the newborn infant (4-6). More recently, interest has developed in other factors in colostrum and milk, such as epidermal growth factor (7, 8), interferon (9, 1 O), and prostaglandins ( 1 1, 12), which can influence cell growth and differentiation in vitro. It is possible that these growth factors and lymphokines in milk may actively enhance the development of the immune system of the breastfed infant, although an in vivo role for milk lymphokines has not yet been demonstrated. To investigate whether these lymphokines might be having an effect in vivo on the immune system of the breast-fed infant, we have studied the development of immune responses in breast-and bottle-fed infants sequentially from 6 days to 9 months of age.Mononuclear cell proliferation in vitro has frequently been used to assess ...