Updated project metadata. The sexually transmitted pathogen Neisseria gonorrhoeae releases outer membrane vesicles (OMVs) during infections. OMVs traffic the major porin PorB, other membrane proteins and lipo-oligosaccharide (LOS) into host innate immune cells and activate programmed cell death pathways and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether this affects immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, included major outer membrane, periplasm, cytoplasmic membrane proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of major outer membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as Il-1andIl-1. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize OMVs to release major proteins and lipids, which affects innate immune responses.