As many new biologically active chemokines have been cloned exploring the genomic DNA sequence database in the vicinity of already known chemokine sequences without demonstrating their natural origin, it is important to transfer findings from in vitro experiments with chemokines into the in vivo situation. With respect to eosinophils and fibroblasts that play an important part in the pathogenesis of allergic and autoimmune diseases, the role of the recently discovered members of the eotaxin family, eotaxin-2 and eotaxin-3, is not really understood. In order to elucidate the origin and biologic potency of the eotaxin family this study was performed. Conventional reverse transcription-polymerase chain reaction analysis was suitable to detect mRNA for eotaxin and eotaxin-3 but not for eotaxin-2 in dermal fibroblasts. In contrast to conventional reverse transcription-polymerase chain reaction, LightCycler analysis revealed that dermal fibroblasts constitutively expressed mRNA not only for eotaxin and eotaxin-3 but also for eotaxin-2. Moreover, with this technique we investigated mRNA expression levels after stimulation of fibroblasts with interleukin-4 and interleukin-4 plus tumor necrosis factor-alpha: the rank order of expression levels within the eotaxin family was eotaxin > eotaxin-3 > eotaxin-2. To address the question of the efficacy of eotaxin-3, we compared its activity with eotaxin, eotaxin-2, monocyte chemotactic protein-3, monocyte chemotactic protein-4, and RANTES in different test systems for eosinophils. The efficacy of the CC chemokines at equimolar concentrations with respect to the chemotactic response of human eosinophils was eotaxin-3 = eotaxin = eotaxin-2 > RANTES > monocyte chemotactic protein-4. The rank order of activity with respect to actin polymerization and release of toxic reactive oxygen species was eotaxin-3 = eotaxin = eotaxin-2 and eotaxin = eotaxin-2 > eotaxin-3 = monocyte chemotactic protein-3 = monocyte chemotactic protein-4 = RANTES, respectively. This study indicated a distinct profile in expression levels of the members of the eotaxin family in dermal fibroblasts. Indeed, all three eotaxin ligands demonstrated activation of human eosinophils with similar efficacies for chemotaxis, cytoskeletal rearrangements, activation of Gi proteins and transients of [Ca2+]i, but a distinct profile of activity with respect to the binding to CCR3 and the release of toxic reactive oxygen species. These findings may help to understand further the role of CC chemokines in fibroblast/eosinophil activation, which is of interest particularly in allergic and autoimmune diseases.