We have investigated reponses to extracellular ATP in human fibroblasts obtained by skin biopsies. Our data show that these cells express a P2X₇ purinergic receptor, as judged by (1) RT-PCR with specific primers, (2) reactivity with a specific anti-P2X₇ antiserum, (3) activation by the selective P2X agonist benzoylbenzoylATP and (4) stimulation of transmembrane ion fluxes. Stimulation with benzoylbenzoylATP, and to a lesser extent with ATP, also caused striking morphological changes and increased formation of cytoplasmic microvesicles. These changes were fully reversible upon nucleotide removal. Two known blockers of P2X receptors, oxidised ATP and pyridoxalphosphate-6-azophenyl-2´,4´disulfonic acid, inhibited the morphological changes fully and the ion fluxes partially. The residual rise in intracellular Ca²⁺ levels and membrane depolarization observed in the presence of the inhibitors were dependent upon activation of a P2Y-type receptor exhibiting a peculiar pharmacological profile, in that CTP was the preferred agonist. ATP stimulation triggered release of the pro-inflammatory cytokine IL-6 in fibroblasts pre-treated with PMA and bacterial endotoxin. These observations reveal a novel pathway for fibroblast activation and for their recruitment in the inflammatory response.