Alveolar Macrophages (AMs) are a subset of tissue macrophages situated in the alveolar milieu. In comparison to their precursor blood monocytes, AMs exhibit distinct physiological functions unique to their anatomic location. However, the molecular details that control monocyte differentiation into AMs remain unknown. This study employed a proteomic approach to define protein characteristics that distinguish AMs from monocytes. AMs and monocytes were obtained from six non-smoking, healthy donors. Whole cell lysates from each donor's AMs and monocytes were analyzed by Two-Dimensional (2D) gel electrophoreses. The protein density for each protein spot in a 2D gel was compared between these two cell-types. Proteins that demonstrated consistent level changes of greater than 2.5-fold in all six donors were subjected to tandem mass spectrometry for protein identity. Using this process, we revealed proteome changes in AMs that relate to their physiological roles in proteolysis, actin re-organization, and cellular adaptation in the unique alveolar milieu. By comparison, blood monocytes displayed higher levels of the proteins involved in transcription, metabolism, inflammation, and in the control of proteolysis. These results provide new insights into the biology of mononuclear phagocytes and set a basis for future causality studies.