Hsp110 proteins constitute a heterogeneous family of abundant molecular chaperones, related to the Hsp70 proteins and exclusively found in the cytosol of eukaryotic organisms. Hsp110 family members are described as efficient holdases, preventing the aggregation and assisting the refolding of heat-denatured model substrates in the presence of Hsp70 chaperones and their co-chaperones. To gain more insights into the mode of action of this protein family we compared two homologues representing two subtypes of Hsp110 proteins, S. cerevisiae Sse1 and H. sapiens Apg-2, in their structural and functional properties in vitro. In contrast to previous publications both proteins exhibited intrinsic ATPase activities, which only in the case of Sse1 could be stimulated by the Hsp40 co-chaperone Sis1. Similar to Hsp70 proteins ATP binding and hydrolysis induced conformational rearrangements in both Hsp110 proteins as detected by tryptophane fluorescence. However, nucleotide induced changes in the proteolytic digestion pattern were detected only for Sse1. Sse1 and Apg-2 thus show significant differences in their biochemical properties, which may relate to differences in their functional roles in vivo.