Alveolar surfactant modulates the antimicrobial function of bronchoalveolar macrophages (BAM). Little is known about the effect of surfactant-associated proteins in bronchoalveolar lavage fluid (BALF) on the interaction of BAM and Blastomyces dermatitidis. We investigated BALF enhancement or inhibition of TNF-α production by BAM stimulated by B. dermatitidis. BAM from CD-1 mice were stimulated with B. dermatitidis without or with normal BALF, surfactant protein A-deficient (SP-A-/-) or surfactant protein D-deficient (SP-D-/-) BALF, or a mixture of SP-A-/- and SP-D -/- BALF. An enzyme-linked immunosorbent assay was used to measure tumor necrosis factor alpha (TNF-α) in culture supernatants. BALFs were standardized in protein concentration. BAM plus B. dermatitidis (BAM-B. dermatitidis) TNF-α production was inhibited ≥47% by BALF or SP-A -/- BALF (at 290 or 580 μg of protein/ml, P < 0.05 to 0.01); in contrast, SP-D-/- BALF did not significantly inhibit TNF-α production. If SP-A-/- BALF was mixed in equal amounts with SP-D -/- BALF, TNF-α production by BAM-B. dermatitidis was inhibited (P < 0.01). Finally, pure SP-D added to SP-D-/- BALF inhibited TNF-α production by BAM-B. dermatitidis (P < 0.01). B. dermatitidis incubated with BALF and washed, plus BAM, stimulated 63% less production of TNF-α than did unwashed B. dermatitidis (P < 0.05). SP-D was detected by anti-SP-D antibody on BALF-treated unwashed B, dermatitidis in an immunofluorescence assay (IFA). The BALF depleted by a coating of B. dermatitidis lost the ability to inhibit TNF-α production (P < 0.05). 1,3-β-Glucan was a good stimulator of BAM for TNF-α production and was detected on B. dermatitidis by IFA. β-Glucan incubated with BALF inhibited the binding of SP-D in BALF to B. dermatitidis as demonstrated by IFA. Our data suggest that SP-D in ?? BALF binds β-glucan on B, dermatitidis, blocking BAM access to β-glucan, thereby inhibiting TNF-α production. Thus, whereas BALF constituents commonly mediate antimicrobial activity, B. dermatitidis may utilize BALF constituents, such as SP-D, to blent the host defensive reaction; this effect could reduce inflammation and tissue destruction but could also promote disease.