1 In order to understand mechanisms that limit the safe ischaemic time of donor hearts, this study evaluated NO/cyclic GMP biosignalling in the recovery of function after cardioplegia and hypothermic storage. 2 Hearts removed from anaesthetized rats were either perfused in working mode (Fresh) or arrested (St. Thomas' II cardioplegia) and stored at 3 degrees C for 8 h (CPL) prior to working mode perfusion. LV work and indices of the production of NO (Ca2+-dependent and Ca2+-independent NOS), cyclic GMP (soluble guanylyl cyclase (sGC) and GTP) and superoxide (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)) were measured. 3 Relative to Fresh hearts, CPL hearts were deficient in cyclic GMP and had poor function. Correction of cyclic GMP deficiency (SNP, 200 microM) improved LV work and LV compliance. SNP effects were prevented by inhibition of sGC (ODQ, 3 microM), and potentiated by inhibition of cyclic GMP-dependent phosphodiesterase (zaprinast, 20 microM). SNP (200 microM) had no effect on function of Fresh hearts. 4 NOS activities (pH = 7.2) were similar in CPL and Fresh hearts, but at end-ischaemic pH (6.3), Ca2+-dependent NOS activity was reduced. The sensitivity of sGC to SNP was greater, and activities of XO and XDH were higher, in CPL than in Fresh hearts. 5 The deficiency in NO biosignalling in CPL hearts may arise due to acidosis-induced inhibition of NOS activity, reduced availability of GTP and/or enhanced inactivation of NO by superoxide. These findings provide rationales for novel strategies to prevent the deficiency in NO biosignalling and so improve the function of the transplanted heart.