Extracellular nucleotides and nucleosides are important signaling molecules in the lung. Nucleotide and nucleoside concentrations in alveolar lining fluid are controlled by a complex network of surface ectonucleotidases. Previously, we demonstrated that influenza A/WSN/33 (H1N1) virus resulted in increased levels of the nucleotide ATP and the nucleoside adenosine in bronchoalveolar lavage fluid (BALF) of wild-type (WT) C57BL/6 mice. Influenza-induced acute lung injury (ALI) was highly attenuated in A₁-adenosine receptor-knockout mice. Because AMP hydrolysis by the ecto-5'-nucleotidase (CD73) plays a central role in and is rate-limiting for generation of adenosine in the normal lung, we hypothesized that ALI would be attenuated in C57BL/6-congenic CD73-knockout (CD73-KO) mice. Infection-induced hypoxemia, bradycardia, viral replication, and bronchoconstriction were all moderately increased in CD73-KO mice relative to WT controls. However, post-infection weight loss, pulmonary edema, and parenchymal dysfunction were not altered. Treatment of WT mice with the CD73 inhibitor 5'-(α,β-methylene) diphosphate (APCP) also had no effect on infection-induced pulmonary edema but modestly attenuated hypoxemia. BALF from both CD73-KO mice and APCP-treated WT mice contained more IL-6 and CXCL-10/IP-10, less CXCL-1/KC, and fewer neutrophils than BALF from untreated WT controls. BALF from APCP-treated WT mice also contained less alveolar macrophages and more TGF-β than untreated WT BALF. These results indicate that CD73 is not necessary for development of ALI following influenza A virus infection, and suggest that tissue non-specific alkaline phosphatase may be responsible for increased adenosine generation in the infected lung. However, they do suggest that CD73 has a previously unrecognized immunomodulatory role in influenza.