In this study we compared the activation of monocytes by different bacterial products via Toll‐like receptors (TLR), and by different proinflammatory mediators. In response to TLR‐2, ‐4 and ‐5 engagement, ∼50% of monocytes produced TNF‐α, compared to only 5% after induction with IFN‐γ or GM‐CSF. Furthermore, a small proportion of monocytes produced IL‐10 after stimulation via TLR, but not after stimulation with cytokines. Both TLR‐ligands and inflammatory cytokines induced the expression of CD25, CD69, CD80 and, surprisingly, also of CD83, commonly regarded as an activation marker for mature dendritic cells (DC). Conversely, TLR‐ligands downregulated CD38, CD86 and ICOS‐L. Importantly, signaling lymphocytic activation molecule (SLAM; CD150) was identified as a monocyte activation marker that could be induced ex novo via TLR‐2, ‐4 and ‐5, but not by single stimulation with monocyte activators like IL‐1, TNF‐α, IFN‐β, IFN‐γ, GM‐CSF or CD40‐L. SLAM expression was transient and required mitogen activated protein kinase (MAPK) p38, but not ERK or JNK, and was surprisingly independent of NF‐κB. SLAM+ monocytes, which are absent in blood, were detected in spleen and tonsils, where they could be localized to T‐cell areas and germinal centers. Together, by comparing the response of monocytes to TLR‐ligands and inflammatory cytokines, we have identified a monocyte activation marker, SLAM, which differs in its inducibility from other monocyte activation markers. SLAM+ monocytes and macrophages were identified for the first time in vivo. Their presence might be a sign of innate immune activation.