In this work, we identified Hsp70 as a novel target of the Sgt1 protein. Using co-immunoprecipitation, affinity chromatography and ELISA we showed that, besides Hsp90, Sgt1 interacts with the heat shock protein, Hsp70. We also found that a deletion mutant of Sgt1, devoid of the C-terminal region, did not bind to either Hsp70 or Hsp90 proteins. Overexpression of S100A6, a calcium binding protein that interacts with the C-terminal part of Sgt1, decreased the amount of chaperone bound to Sgt1. However, the effect of S100A6 on this interaction was not observed in BAPTA/AM treated cells in which Ca(2+) level was decreased. This suggests that the interaction of Sgt1 with Hsp70 and Hsp90 is regulated by S100A6 in a Ca(2+)-dependent manner.