The CD16⁺ subset of peripheral blood monocytes (Mo) is expanded dramatically during inflammatory conditions including sepsis, HIV-1 infection, and cancer. CD16⁺ express high levels of CX3CR1, which mediates arrest onto CX3CL1-expressing endothelial cells (EC) under flow conditions. In contrast, attachment of CD16⁻ Mo onto cytokine-activated EC is independent of CX3CL1. Here, we investigate the ability of CD16⁺ and CD16⁻ Mo to produce proinflammatory cytokines upon interaction with CX3CL1-expressing HUVEC. We demonstrate that CD16⁺ but not CD16⁻ Mo produce high levels of IL-6, CCL2, and matrix metalloproteinase (MMP)-9 when cocultured with TNF/IFN-γ-activated HUVEC or nonactivated HUVEC expressing CX3CL1. Furthermore, supernatants from Mo cocultured with cytokine-activated HUVEC induce neuronal death in vitro. These results suggest that membrane-bound CX3CL1 stimulates production of IL-6, CCL2, and MMP-9 by CD16⁺ Mo, likely via engagement of CX3CR1. Thus, expansion of CD16⁺ Mo and their accumulation onto CX3CL1-expressing EC may result in recruitment of Mo and T cell subsets at sites of inflammation in response to CCL2, IL-6-induced cell activation and/or differentiation, and MMP-9-mediated vascular and tissue injury.