Mouse Hsp70 (70 kDa heat shock protein) is preferentially induced by heat or stress stimuli. We previously found that Hsp70 is constitutively expressed in A6 mouse mesoangioblast stem cells, but its possible role in these cells and the control of its basal transcription remained unexplored. Here we report that in the absence of stress, Ku factor is able to bind the HSE (heat shock element) consensus sequence in vitro, and in vivo it is bound to the proximal hsp70 promoter. In addition, we show that constitutive hsp70 transcription depends on the co-operative interaction of different factors such as Sp1 (specificity protein 1) and GAGA-binding protein with Ku factor, which binds the HSE consensus sequence. We used mRNA interference assays to select knockdown cell clones. These cells were able to respond to heat stress by producing a large amount of Hsp70, and produced the same amount of Hsp70 as that synthesized by stressed A6 cells. However, severe Hsp70 knockdown cells had a longer duplication time, suggesting that constitutive Hsp70 expression has an effect on the rate of proliferation.