The evaluation of monocytes recruited into the alveolar space under both physiological and inflammatory conditions is hampered by difficulties in discriminating these cells from resident alveolar macrophages (rAMs). Using the intravenous injected fluorescent dye PKH26, which accumulated in rAMs without labeling blood leukocytes, we developed a technique that permits the identification, isolation, and functional analysis of monocytes recruited into lung alveoli of mice. Alveolar deposition of murine JE, the homologue of human monocyte chemoattractant protein (MCP)-1 (JE/MCP-1), in mice provoked an alveolar influx of monocytes that were recovered by bronchoalveolar lavage and separated from PKH26-stained rAMs by flow cytometry. Alveolar recruited monocytes showed a blood monocytic phenotype as assessed by cell surface expression of F4/80, CD11a, CD11b, CD18, CD49d, and CD62L. In contrast, CD14 was markedly upregulated on alveolar recruited monocytes together with increased tumor necrosis factor-α message, discriminating this monocyte population from peripheral blood monocytes and rAMs. Thus monocytes recruited into the alveolar air space of mice in response to JE/MCP-1 keep phenotypic features of blood monocytes but upregulate CD14 and are “primed” for enhanced responsiveness to endotoxin with increased cytokine expression.