The phenomenon of induced tolerance to a normally lethal episode of ischaemia by preconditioning with sub-lethal ischaemia has been linked to induction of the 72 kDa heat-shock protein (HSP72). However, a direct correlation between HSP72 expression and ischaemic preconditioning in vivo has not been proven. Using an in vitro model of ischaemia-related neuronal damage we have investigated whether HSP72 protein expression is temporally correlated with subsequent tolerance to a normally lethal ischaemic episode. Organotypic hippocampal slice cultures were maintained in vitro for 14 days before being exposed to hypoxia for 15-180 min. Periods of hypoxia shorter than 60 min did not produce neuronal damage. No HSP72 immunoreactivity was observed in either untreated cultures or in those exposed to hypoxia for 15 min. Following 30 and 45 min hypoxia a significant induction of HSP72 occurred in neurons of both the CA1 and CA3/4 regions of the pyramidal cell layer. A significant number of microglia were positively stained with HSP72. The peak of HSP72 expression occurred 18 h after the induction of hypoxia but remained significantly elevated for 48 h post-hypoxia. Prolonged hypoxia (60 or 180 min) produced a selective lesion of the CA1 pyramidal cell layer which was not associated with an induction of HSP72. Pre-conditioning with 45 min hypoxia 18 h prior to 180 min hypoxia did not reduce the neuronal damage associated with 180 min hypoxia alone. These data strongly suggest that HSP72 does not directly confer tolerance in this in vitro model of ischaemia-related neuronal death.