Neutrophils use intricate mechanisms for capturing and killing invading microorganisms. One mechanism entails the release of relaxed chromatin from the cell. Microbes are trapped by the extracellular chromatin and exposed to high local concentrations of bactericidal compounds. We examine the regulation of chromatin release by testing the contribution of microtubules and the actin cytoskeleton to the deployment of neutrophil extracellular traps (NETs). Incubation of human neutrophils with nocodazole, a tubulin polymerization inhibitor, or cytochalasin D, an inhibitor of actin filamentation, severely diminished the ability of neutrophils to respond to LPS by releasing chromatin from the cells. In addition, pretreatment of neutrophils with M1/70, a monoclonal antibody to the Mac-1 integrin adhesion receptor, drastically reduced the deployment of chromatin into NETs. Analysis of histone deimination, the conversion of arginine to citrulline in 3 of the 4 core histones by peptidylarginine deiminase 4, revealed that the treatments inhibiting NET formation also reduced histone deimination. Our data indicate that NET formation requires functional tubulin and actin filaments and responds to engagement of Mac-1 integrins. Because histone deimination coincides with the release of NETs, we propose that these events represent overlapping mechanisms of neutrophil responses to infections.