Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na(+)/H(+)-exchanger (NHE1) and Na(+),K(+),2Cl(-)-cotransporter (NKCC1) to RVI. Hypertonic stress induced caspase-3 activity in WEHI cells and iMEFs, an effect potentiated by Hsp70 in WEHI cells but inhibited by Hsp70 in iMEFs. Osmotic shrinkage-induced PCD was associated with Hsp70-inhibitable cysteine cathepsin release in iMEFs and attenuated by caspase and cathepsin inhibitors in WEHI cells. Treatment with TNF-alpha or the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) reduced the viability of WEHI cells further under isotonic and mildly, but not severely, hypertonic conditions. Thus, it is concluded that shrinkage-induced PCD involves both caspase- and cathepsin-dependent death mechanisms and is potently counteracted by Hsp70.