Paired samples of milk and serum collected 3 days postpartum from 20 women were tested for the presence and level of interleukin (IL)-1 beta, 1L-6, IL-12, tumor necrosis factor alpha(TNF-alpha), and interferon-gamma (IFN-gamma) by enzyme immunoassay, The expression of these cytokine mRNAs in milk macrophages from eight donors were semiquantitatively analyzed by reverse transcriptase-polymerase chain reaction, The effects of respiratory syncytial virus (RSV) infection on cytokine production were determined in five samples of milk macrophages. Over 90% of the milk samples tested exhibited detectable levels of IL-1 beta, 1L-6, and TNF-alpha, No 1L-12 or IFN-gamma activity was detected in the milk, IL-6 activity was weakly detected in about 45%, and TNF-alpha activity in about 10% of the serum samples tested, However, no IL-1 beta, 1L-12, or IFN-gamma activity was demonstrated in any of the serum samples, Milk macrophages from eight subjects all exhibited mRNA for IL-1 beta, TNF-alpha, and IL-6, and lFN-gamma mRNA in six of eight subjects, although no IFN-gamma was detected in any of the 20 samples of milk tested, RSV exposure resulted in a 2- to 100-fold increase in the expression of IL-1 beta, 1L-6, and TNF-a mRNA as well as cytokine protein, Although RSV infection enhanced the expression of IFN-gamma mRNA, no detectable IFN-gamma was produced by the milk macrophages, These observations suggest that the milk macrophages are actively engaged in the physiological production of IL-1 beta, 1L-6, TNF-alpha, and IFN-gamma in the mammary gland and continue to possess the capacity to increase production of these cytokines in response to RSV and possibly other viral infections.