Pulmonary surfactant is inactivated in meconium aspiration syndrome and neonatal pneumonia. Development of an exogenous surfactant less sensitive to inactivation might be useful for treating these diseases. We investigated in vitro whether addition of the cationic cyclic membrane cross-linking peptide polymyxin B (PxB) and/or calcium chloride (CaCl2) to modified porcine surfactant Curosurf increases resistance to meconium-induced inactivation of surface activity while antimicrobial activity of PxB is maintained. To study bacterial proliferation, Escherichia coli, group B streptococci (GBS), or Staphylococcus aureus were incubated 0-5 h in saline or in meconium in the presence or absence of Curosurf with or without PxB. PxB and CaCl2 improved spreading and adsorption of Curosurf. Curosurf plus CaCl2/PxB needed a 4-fold increase of meconium concentration to increase dynamic surface tension significantly compared with Curosurf plus CaCl2 alone, indicating that PxB further increases the resistance of Curosurf to meconium-induced inactivation. Meconium alone like meconium/Curosurf promoted growth of E. coli and GBS, but addition of Curosurf/PxB or PxB alone significantly reduced the growth of E. coli. Biophysical and antibacterial properties of Curosurf and PxB may be combined into a useful adjunct in the treatment of neonatal Gram-negative pneumonia and/or meconium aspiration syndrome.