Stress-activated protein kinases (SAPKs) or c-Jun amino-terminal kinases (JNKs), which belong to a subgroup of the mitogen-activated protein kinase (MAPK) superfamily, are activated in response to a variety of stresses in mammalian cells. An activity to activate a recombinant rat SAPK alpha was detected in extracts obtained from rat fibroblastic 3Y1 cells exposed to hyperosmolar media and was resolved into unadsorbed and adsorbed fractions on Q-Sepharose chromatography. The adsorbed activity was identified as XMEK2/SEK1/MKK4 by using several anti-XMEK2 antibodies. Thus, a 45-kDa protein that was recognized specifically by these anti-XMEK2 antibodies co-eluted with the SAPK alpha activating activity during chromatography on Q-Sepharose and Superose 6, and the activity could be immunoprecipitated by the antibodies from these fractions. The unadsorbed activity, whose level was much greater than that of the adsorbed activity, did not contain XMEK2/SEK1/MKK4 and was also activated in a time-dependent manner by osmotic shock. This activity was further resolved into several peaks during chromatography on heparin-Sepharose and hydroxylapatite. Most of these peaks eluted separately from major peaks of a kinase activity toward p38/MPK2, another subgroup of the MAPK superfamily, whereas the activated XMEK2/SEK1/MKK4 could phosphorylate p38/MPK2 efficiently. These results indicate the existence of multiple activators for SAPK/JNK; one is XMEK2/SEK1/MKK4, and the others are previously undescribed factors.