Traditionally, the identification and quantification of eosinophils in inflammatory tissues and exudates has been primarily based upon morphologic criteria and manual counting. In this study, we describe a new flow cytometry-based assay to enumerate eosinophils present in murine bronchoalveolar lavage fluid (BAL) and lung parenchyma obtained from the normal/non-inflamed respiratory tract, following experimentally-induced allergic pulmonary inflammation, and during experimental infection with respiratory syncytial virus (RSV). By using a murine Siglec-F-specific antibody in combination with antibodies directed to CD45 and CD11c, we demonstrate that eosinophils can be distinguished from other cell types in the BAL fluid and lung parenchyma based upon their distinct CD45(+) Siglec-F(+) and CD11c(low/-) staining profile. In the BAL fluid, this flow cytometry-based method of eosinophil identification/quantitation yields results comparable to the standard morphology-based method without the potential observer bias or staining artifacts inherent in morphology-based quantitation. Furthermore, this flow cytometry-based method can be directly adapted to enumerate eosinophils infiltrating the inflamed lung parenchyma, thereby obviating the need for quantitative morphometry of tissue sections.