Abstract2—5A Synthetase and 2—5A phosphodiesterase were determined by analytical capillary isotachophoresis in comparison to radioenzymatic methods. By means of isotachophoretic analysis, a frequently used radioenzymatic 2—5A synthetase assay was optimized and the results of both assays were compared. Using the isotachophoretic assay the influence of interferon‐related cytokines (tumor necrosis factor‐alpha and interleukine‐2) on 2—5A synthetase induction in neuroblastoma cells was estimated. In contrast to mononuclear blood cells, the tumor necrosis factor induced 2—5A synthetase in these cells. 2—5A Phosphodiesterase was determined using an isotachophoretic assay and a radioenzymatic method. Degradation of A2′p5′A2′p5′A (trimeric form of 2—5A core) was measured by isotachophoresis whereas degradation of a mixture of phosphorus‐32 labeled 2—5A cores was registrated by radioenzymatic assay. Activity of 2—5A phosphodiesterase was only insignificantly enhanced by interferon in mononuclear blood and neuroblastoma cells. In contrast to the radioenzymatic assays, an accurate determination of 2—5A synthetase as well as of 2—5A phosphodiesterase is possible using the isotachophoretic method because the reactions are followed by measuring the substrate