Pneumonia and systemic infection are common in premature infants. The antimicrobial peptides human beta-defensin 1 and 2 (hBD-1 and hBD-2) and the cathelicidin LL-37/hCAP-18 are effector molecules of the innate respiratory immune system. It is unknown whether these host defense substances are produced in the respiratory tract of newborns. Concentrations of these peptides were determined in tracheal aspirates of mechanically ventilated newborn infants. All three antimicrobial peptides could be detected in airway lining fluid with equivalent levels in term and preterm newborns. Concentrations of antimicrobial peptides correlated with each other and with levels of interleukin-8 and tumor necrosis factor-alpha in the bronchoalveolar lavage fluid. Pulmonary or systemic infections were associated with significantly increased concentrations of LL-37, hBD-1, and hBD-2. Western blotting detected mature peptides in the lavage fluid. In conclusion, mucosal antimicrobial peptides are present in lung secretions of premature and mature newborns. The molecules are upregulated in response to infection and inflammation and probably represent effector molecules of the respiratory defense system.