Summary Arabidopsis thaliana contains 18 genes encoding Hsp70s. This heat shock protein superfamily is divided into two sub‐families: DnaK and Hsp110/SSE. In order to functionally characterize members of the Hsp70 superfamily, loss‐of‐function mutants with reduced cytosolic Hsp70 expression were studied. AtHsp70‐1 and AtHsp70‐2 are constitutively expressed and represent the major cytosolic Hsp70 isoforms under ambient conditions. Analysis of single and double mutants did not reveal any difference compared to wild‐type controls. In yeast, SSE protein has been shown to act as a nucleotide exchange factor, essential for Hsp70 function. To test whether members of the Hsp110/SSE sub‐family serve essential functions in plants, two members of the sub‐family, AtHsp70‐14 and AtHsp70‐15, were analysed. Both genes are highly homologous and constitutively expressed. Deficiency of AtHsp70‐15 but not of AtHsp70‐14 led to severe growth retardation. AtHsp70‐15‐deficient plants were smaller than wild‐type and exhibited a slightly different leaf shape. Stomatal closure under ambient conditions and in response to ABA was impaired in the AtHsp70‐15 transgenic plants, but ABA‐dependent inhibition of germination was not affected. Heat treatment of AtHsp70‐15‐deficient plants resulted in drastically increased mortality, indicating that AtHsp70‐15 plays an essential role during normal growth and in the heat response of Arabidopsis plants. AtHsp70‐15‐deficient plants are more tolerant to infection by turnip mosaic virus. Comparative transcriptome analysis revealed that AtHsp70‐15‐deficient plants display a constitutive stress response similar to the cytosolic protein response. Based on these results, AtHsp70‐15 is likely to be a key factor in proper folding of cytosolic proteins, and may function as nucleotide exchange factor as proposed for yeast.