Conversion of the highly surface-active subtype of pulmonary surfactant known as large surfactant aggregates (LA) to small aggregates (SA) with poor surface activity has recently been shown to occur upon cyclic changes of the air-liquid interface area in vitro. By subjecting pooled rabbit bronchoalveolar lavage fluid (BALF) to this maneuver, we found that conversion of LA to SA was accompanied by a marked decline in the ability of the remaining LA fraction to reduce surface tension by adsorption and during film compression on a pulsating bubble surfactometer. SA obtained by centrifugation of noncycled rabbit BALF had a similar phospholipid (PL) but different neutral lipid (NL) composition than did the LA. Upon cycling, the increased formation of SA obliterated this difference. No substantial difference in the PL, NL, or fatty acid profile of LA was noted before and after cycling. In contrast, the content of surfactant apoprotein-B (SP-B) in the LA decreased dramatically to nearly undetectable levels during the cycling maneuver, and this decline in SP-B content was closely correlated with the decrease in proportional appearance of LA and loss of surface activity of this fraction. Reconstitution of LA with intact SP-B after cycling virtually fully restored the surface activity of this surfactant subtype. When comparing lavage samples from adults with acute respiratory distress syndrome (ARDS; n = 10) with samples from healthy controls (n = 11), we noted a marked reduction of SP-B in the LA fraction. There was a significant correlation between the SP-B content of the LA fraction and the relative percentage of LA in BALF or the lower surface activity of this surfactant subtype. We conclude that an SP-B-related loss of LA integrity and function may substantially contribute to the decline of this surfactant subtype and the loss of its surface activity during cycling in vitro and in clinical ARDS.