We examined changes in proteinase activities in P19 embryonal carcinoma cells during retinoic acid-induced differentiation. The interleukin-1 beta converting enzyme (ICE)-like Ac-YVAD-MCA hydrolytic activity was increased about 6-fold by treatment with retinoic acid. This activity was inhibited by N-ethylmaleimide and Ac-YVAD-H but not by E-64, EDTA, PMSF, or amastatin. The ICE-like activity in P19 cells eluted as a single peak just after the void volume on gel filtration. No ICE-like activity was observed at a molecular mass of 30-50 kDa. Enzymatic purification, Western blot analysis, and an immunoabsorption study demonstrated that the ICE-like activity in P19 cells is caused by the proteasome, and is stimulated during retinoic acid-induced differentiation. The proteasome purified from mouse liver also cleaved Ac-YVAD-MCA. These results strongly suggest that the proteasome is a major ICE-like proteinase in P19 cells and may be involved in the neural differentiation and the apoptotic pathway.