Macrophage‐derived chemokine (MDC)/CCL22 is a CC chemokine active on dendritic cells (DC), NK cells and Th2 lymphocytes. The present study was aimed at comprehensively investigating MDC production in vitro and in vivo. DC were the most potent producers of MDC among leukocytes tested. Endothelial cells did not produce MDC under a variety of conditions. Signals that induce maturation (lipopolysaccharide, IL‐1, TNF, CD40 ligand, recognition of bacteria and yeast) dramatically augmented MDC production, and dexamethasone and vitamin D3 blocked it. Prostaglandin E₂, which blocked the acquisition of IL‐12 production and the capacity to promote Th1 generation, did not affect MDC production. Using mass spectrometry‐based techniques, DC supernatants were found to contain N‐terminally truncated forms of MDC [MDC(3–69), MDC(5–69) and MD(C7–69)] as well as the full‐length molecule. In vivo, CD1a⁺, CD83⁺, MDC⁺ DC were found in reactive lymph nodes, and in Langerhans' cell histiocytosis. Skin lesions of atopic dermatitis patients showed that CD1a⁺ or CD1b⁺ DC, and DC with a CD83⁺ phenotype were responsible for MDC production in this Th2‐oriented disorder. Thus, DC are the predominant source of MDC in vitro and in vivo under a variety of experimental and clinical conditions. Processing of MDC to MDC(3–69) and shorter forms which do not recognize CCR4 is likely to represent a feedback mechanism of negative regulation.