Pulmonary surfactant isolated from a number of animal species contains a protein of molecular weight 38,000 and several very hydrophobic proteins soluble in solvents often used to extract lipids. Although found to be intimately associated with surfactant lipids, localization of the very hydrophobic proteins to alveolar epithelial type II cells, the cells involved in the synthesis and secretion of pulmonary surfactant, has not been demonstrated. Hydrophobic proteins were extracted along with lipids from human surfactant, and, after extraction with deoxycholate, were used to raise an antiserum. The antiserum was characterized by the immunoblotting ("Western") technique, using blots of pulmonary surfactant, and of hydrophobic proteins prepared by deoxycholate extraction and by Sephadex LH-20 chromatography. The antiserum shows reactivity to a 6.5- to 18-kDa surfactant-associated hydrophobic protein, which appears to be the major component of deoxycholate extracted proteins, and has Phe as the N-terminal amino acid. In addition, 28-kDa, 40-kDa, 50-kDa, and 70-kDa bands (chemically unreduced) are seen in human pulmonary surfactant. Whether these bands represent precursors of the major surfactant-associated hydrophobic proteins, different proteolytic cleavage products of the precursor protein, distinct proteins, or associated forms of the same protein is not yet clear. When used in the immunoperoxidase staining of human lung sections, the antiserum yielded a staining pattern identical to that obtained by the use of an antiserum to the 35-kDa surfactant protein. The number, size, and location of the stained cells are consistent with their being the alveolar epithelial type II cells.