Neutrophils are front-line-defense cells of the innate immune system and a source of reactive oxygen species, inflammatory cytokines, lipid mediators, and tissue-damaging enzymes. They play important roles in both the goblet cell metaplasia in chronic bronchitis and the destruction of lung tissue in emphysema and thus are one of the primary effector cell types in COPD. In order to investigate the mechanism of neutrophil activation by cigarette smoke, neutrophils isolated from peripheral blood of human non-smokers were treated in vitro either directly with cigarette smoke-bubbled phosphate-buffered saline (SBPBS) or indirectly with supernatants from SBPBS-treated MonoMac-6 (mm6) cells. Readout parameters were changes in expression of the surface markers CD11B, CD66B, and CD62L; release of the granule proteins matrix metalloproteinase 9, matrix metalloproteinase 8, and lactoferrin; and oxidative burst induction as measured by n-formyl-methionyl-leucyl-phenylalanine-mediated superoxide release. Both direct and indirect treatment with SBPBS resulted in the priming of neutrophils as measured by an increase in CD11B and CD66B, a reduction in CD62L, and the release of granule proteins. An increase in the oxidative burst response was seen with indirect treatment only. Results suggest that cigarette smoking leads to continuous activation of neutrophils, which perpetuates the chronic lung inflammation seen in COPD patients.