Bleomycin is a widely accepted cancer drug but may induce life-threatening interstitial lung disease in a subset of patients. We evaluated the effect of bleomycin administration on pulmonary surfactant function and composition in rabbit lungs. In order to obtain a uniform response to bleomycin, aerosol technology was employed for bronchoalveolar delivery of 1.8 U/kg b.w. bleomycin. On days 4, 8, 16, 24, 32, and 64 after challenge, bronchoalveolar lavages were performed. Sham-aerosolized rabbits served as controls. In the early acute respiratory distress syndrome (ARDS)-like post-bleomycin period (4-16 days), marked loss of surface activity of the large surfactant aggregate (LA) fraction of surfactant was noted. In parallel, reduced percentages of LA, but only minor changes in surfactant apoproteins (SP)-A, SP-B, and SP-C, were encountered. Analysis of the surfactant lipid profile showed impressively enhanced cholesterol and significantly decreased phosphatidylglycerol (PG) levels. The relative content of dipalmitoyl-PC (DPPC) was slightly increased, and a several-fold increase within the 1-O-alkyl-2-acyl subclass of PC was observed. During the prolonged fibroproliferative period, a highly significant downregulation of SP-B and SP-C levels was observed. This was paralleled by an upregulation of the total extracellular phospholipid pool, with a far-reaching normalization of the (phospho)-lipid profile. The biophysical surfactant function never fully normalized within the 64-day observation period. In conclusion, bleomycin caused marked abnormalities of pulmonary surfactant, with the profile of changes being different between the early ARDS and the late fibrotic phase.