To further evaluate the significance of p38 MAPK as trigger or mediator in ischaemic preconditioning, anisomycin and SB 203580 were used to manipulate its activation status. Special attention was given to the concentration of the drugs and protocols used. The isolated perfused rat heart, subjected to either 25 min global ischaemia or 35 min regional ischaemia, was used as experimental model. This was preceded by anisomycin (2 or 5 muM: 3 x 5 min; 5 muM: 5 min or 10 min; 5 muM: 10 min + 10 min washout or 20 muM: 20 min) or SB 203580 (2 muM: 3 x 5 min; before and during 3 x 5 min or 1 x 5 min ischaemic preconditioning; 10 min). Endpoints were functional recovery during reperfusion and infarct size.Anisomycin, regardless of the protocol, reduced infarct size, but did not improve functional recovery. In a number of experiments activation of JNK by anisomycin was blocked by SP 600125 (10 muM). SP 600125 had no effect on the anisomycin-induced reduction in infarct size. SB 203580 when administered for 10 min before sustained ischaemia, improved functional recovery and reduced infarct size. SB 203580 could not abolish the beneficial effects of a multi-cycle preconditioning protocol, but it significantly reduced the outcome of 1 x 5 min preconditioning. In all hearts improved functional recovery and reduction in infarct size were associated with attenuation of p38 MAPK activation during sustained ischaemia-reperfusion. The results indicate that activation of p38 MAPK acts as a trigger of preconditioning, while attenuation of its activation is a prerequisite for improved recovery and a reduction in infarct size.