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High-performance liquid chromatography (HPLC) is useful for the purification and separation of immunoregulatory cytokines, such as macrophage-derived interleukin 1 (IL 1). In addition to macrophages, epidermal cells also release a mediator, epidermal cell (EC) derived thymocyte-activating factor (ETAF), whcih cannot be separated from IL 1. Moreover, it has been shown recently that EC produce a distinct interleukin 3-like mast cell-activating factor (EC IL 3). This study was performed to investigate whether HPLC may be useful for the separation of EC-derived cytokines, such as ETAF and EC IL 3. For factor production, a murine EC line (Pam 212) was used. ETAF activity was measured using the thymocyte costimulator assay. EC IL 3 was was determined by induction of the proliferative activity of an IL 3-dependent cell line (32 DCL). Using a TSK 125 size-exclusion column and phosphate-buffered saline (pH 7.2) as the mobile phase, ETAF was eluted with an apparent molecular weight of 17 kD, and EC IL 3 with a molecular weight of 28 kD. When EC supernatants were chromatofocused on a Mono P column, ETAF activity was eluted with apparent p I values of 6.8, 6.2 and 5.3, and ECL IL 3 activity with p I 7.8, 7.4 and 7.1. When reversed-phase HPLC (RP-HPLC) (equilibration with water and a 0–100% concave acetonitrile gradient) was applied ETAF exhibited four distinct peaks, whereas EC IL 3 was eluted as one major peak between 70 and 80% acetonitrile. Separation on a Bio-Gel HPHT column with a sodium phosphate gradient was not satisfactory, but the recovery was high. It is concluded that chromatofocusing on Mono P and RP-HPLC are suitable methods for the separation of cytokines, such as ETAF and EC IL 3, both of which are produced by EC.