To investigate the function of alveolar macrophages (AM) and the mechanisms of impairment in pulmonary alveolar proteinosis, we established in culture AM from three patients and from eight normal nonsmokers and assessed phagocytosis and phagolysosome fusion by the acridine orange assay with live yeast as the phagocytic challenge. Alveolar macrophages from the patients with pulmonary alveolar proteinosis ingested fewer yeasts per cell than did normal AM (mean +/- SE, 2.3 +/- 0.3 vs 3.3 +/- 0.2; p less than 0.05) and had decreased phagolysosome fusion (33 +/- 6 percent vs 64 +/- 1 percent; p less than 0.001). Alveolar macrophages from three normal subjects were incubated with cell-free fractions isolated by centrifugation of lavage fluid from the patients at 250 g (P1) or centrifugation of P1 supernatant at 20,000 g (P2). The P1 fraction did not decrease the number of AM ingesting yeast or the number of yeast cells ingested per cell, but the P2 fraction decreased both phagocytic indices. Conversely, phagolysosome fusion was depressed by the P1 fraction (48 +/- 3 percent vs 66 +/- 2 percent for untreated AM from the same subject; p less than 0.02) but not by the P2 fraction. Significant morphologic changes were noted in AM cocultured with both P1 and P2. Comparable concentrations of pooled P2 fractions from normal subjects did not decrease phagocytic indices in normal AM. These data confirm that AM in pulmonary alveolar proteinosis are dysfunctional, and, in particular, the finding of decreased phagolysosome fusion may be related to the high incidence of uncommon infections in these patients. We have shown that different fractions of alveolar filling material from patients with pulmonary alveolar proteinosis have unique effects on the phagocytic process in the normal AM, and the induced defects may be associated with apparent uptake of this material. These observations further support the hypothesis that in patients with pulmonary alveolar proteinosis, locally produced "toxic" substances may lead to impaired alveolar clearance and contribute to the pathogenesis of this disease.