Thermoplasma acidophilum cell extracts were fractionated by gel filtration. Proteasomes were eluted as two major peaks. The first one (molecular mass(r) about 2 MDa) contained proteasomes associated with DNA/protein complexes. Proteasomes eluted in the other peak were partially resolved into three subpeaks and based on their preferential hydrolysis of casein, Z-GGL-MCA, and suc-LLVY-MCA, were designated C, L and Y, respectively. Further purification of proteasomes from peak Y resulted in a homogenous enzyme preparation, whereas proteasomes purified from peak C contained a homomultimeric protein composed of 20 kDa subunits. Thus, association of proteasomes with this protein seems to be responsible for the observed increase in molecular mass and for inhibition of caseinolytic activity by Ca2+-ions.