In xenotransplantation long ischemic time of grafts is supposed to have a marked influence on hyperacute rejection (HXR). We investigated the influence of different cold ischemic times on HXR of ex vivo "working pig hearts" perfused with human blood. Xenoreactive natural antibodies (XNAb) as a trigger of HXR were eliminated by Ig-Therasorb immunoadsorption (IA). Explanted Landrace pig hearts of group G1 and group G3 (with additional IA) underwent 4 h of cold ischemia prior to xenoperfusion. Control groups G2 and G4 (with IA) were kept ischemic for only 46.6 +/- 15.8 and 51.2 +/- 4.2 min, respectively. Ischemic time prolonged the perfusion time in our working heart model (G1: 356 +/- 46.1 min; G2: 125 +/- 31 min; P < 0.05). IA had no additional impact on perfusion time but was effective by itself. The heart weight increased fourfold more in G2 as compared to the other groups. IA without ischemia significantly improved cardiac output in G4 (G3: 198.8 +/- 15.4 mL/min; G4: 338.5 +/- 16.0 mL/min). Coronary flow in G2 was significantly lower than in G1 (G1: 157.9 +/- 9.15 mL/min; G2: 59.4 +/- 20.1 mL/min). Histological signs of HXR (light and electron microscopy) could be found in G2 in contrast to the other groups. Parameters of serological damage showed a minimum in G4 and the maximum in G2. In G1 XNAb were nearly equally eliminated immediately after the start of xenoperfusion as in IA groups G4 and G3. Four hours of ischemic time showed beneficial effects in preventing HXR, possibly caused by changes of the endothelial cell surface (for example, glycosylation or loss of alpha1-3Gal epitopes with a hapten effect).