Levels of intracellular multi-ubiquitinated proteins in heat-shocked HeLa cells were investigated using a monoclonal antibody specific to multi-ubiquitin chains. After heat-shock treatment at 42-44 degrees C for 30 min, the level of multi-ubiquitinated proteins increased within the first 2 h at 37 degrees C and returned to the initial level within the following 2 h. The accumulation of multi-ubiquitin conjugates was elevated by increasing the temperature, while the opposite was the case for the level of ubiquitinated histone H2A. Immunocytochemical analysis revealed that the amount of ubiquitin conjugates rapidly increased in the cytosol and concomitantly decreased in the nucleus under heat-shock conditions. The heat-shock treatment elicited little apparent change in the activity of the 26S proteasome, but it did induce a gradual increase in activity of the ubiquitinating enzyme system. These results strongly suggest that the level of cytoplasmic multi-ubiquitinated proteins and that of nuclear ubiquitinated histone H2A increases and decreases, respectively, in response to heat shock and that the heat-shock-induced accumulation of multi-ubiquitinated proteins is caused by activation of the ubiquitinating enzyme system rather than inactivation of the 26S proteasome.