Mice with surfactant protein (SP)-D deficiency have three to four times more surfactant lipids in air spaces and lung tissue than control mice. We measured multiple aspects of surfactant metabolism and function to identify abnormalities resulting from SP-D deficiency. Relative to saturated phosphatidylcholine (Sat PC), SP-A and SP-C were decreased in the alveolar surfactant and the large-aggregate surfactant fraction. Although large-aggregate surfactant from SP-D gene-targeted [(−/−)] mice converted to small-aggregate surfactant more rapidly, surface tension values were comparable to values for surfactant from SP-D wild-type [(+/+)] mice. ¹²⁵I-SP-D was cleared with a half-life of 7 h from SP-D(−/−) mice vs. 13 h in SP-D(+/+) mice. Although initial incorporation and secretion rates for [³H]palmitic acid and [¹⁴C]choline into Sat PC were similar, the labeled Sat PC was lost from the lungs of SP-D(+/+) mice more rapidly than from SP-D(−/−) mice. Clearance rates of intratracheal [³H]dipalmitoylphosphatidylcholine were used to estimate net clearances of Sat PC, which were approximately threefold higher for alveolar and total lung Sat PC in SP-D(−/−) mice than in SP-D(+/+) mice. SP-D deficiency results in multiple abnormalities in surfactant forms and metabolism that cannot be attributed to a single mechanism.