Neutrophils, or polymorphonuclear leukocytes, comprise a crucial component of innate immunity, controlling bacterial and fungal infection through a combination of both oxidative and nonoxidative mechanisms. Indeed, neutrophils are believed to play an important role in controlling infection caused by the major human pathogen Streptococcus pneumoniae. However, the method by which neutrophils kill the pneumococcus as well as other Gram-positive bacteria, is not fully understood. We investigated human neutrophil killing of the pneumococcus in a complement-dependent opsonophagocytic assay. In contrast to other Gram-positive organisms, inhibition of the NADPH oxidase did not affect killing of S. pneumoniae. Supernatant from degranulated neutrophils killed the pneumococcus, suggesting a role for granular products. When neutrophil granule proteases were inhibited with either a protease mixture, or specific serine protease inhibitors 4-(2-Aminoethyl) benzenesulfonylfluoride and diisopropylfluorophosphate, killing by neutrophils was inhibited in a manner that correlated with increased intracellular survival. All three compounds inhibited intracellular activity of the three major neutrophil serine proteases: elastase, cathepsin G, and proteinase 3. Additionally, purified elastase and cathepsin G were sufficient to kill S. pneumoniae in a serine protease dependent-manner in in vitro assays. Inhibition studies using specific inhibitors of these serine proteases suggested that while each serine protease is sufficient to kill the pneumococcus, none is essential. Our findings show that Gram-positive pathogens are killed by human neutrophils via different mechanisms involving serine proteases.