The effects of glutaraldehyde pretreatment and allograft rejection in arterial grafts were assessed, using iso- and allografts in rats. An in situ glutaraldehyde fixation procedure was used to obtain homogeneous cross-linked vascular biografts. Ten Lewis rats were isografted, ten were isografted with a glutaraldehyde-treated aortic segment, ten were allografted with aortic segments from brown Norway (BN) inbred rats, and ten were allografted with glutaraldehyde-treated BN aortas. The macroscopic and microscopic appearances of the grafts were analyzed 3 weeks after the initial surgery. Immunological injury to the media and the intimal response were quantified morphometrically after monochromatic staining of cell nuclei (hematoxylin after periodic acid), elastin (orcein), and calcification (Von Kossa). Untreated isografts were normal. Untreated allografts showed the classical signs of arterial wall rejection: adventitial inflammatory granuloma, reduced medial thickness and smooth muscle cell density, and greatly increased intimal thickness (P < 0.005). Glutaraldehyde treatment significantly decreased the medial thickness in both iso- and allografts (P < 0.001) and prevented the intimal proliferative response (P < 0.005), but did not change adventitial inflammation. It also induced massive calcification mainly in isografts (P < 0.001). Histomorphological modifications of glutaraldehyde-treated grafts are consistent with a partial protective effect of glutaraldehyde against the rejection process, but also with an induction of a nonspecific inflammatory reaction. Glutaraldehyde-induced cross-linking of the extracellular matrix was responsible for ectopic calcification of the arterial grafts which was independent of the rejection process.