The use of biological materials in the construction of bioprostheses requires the application of different chemical procedures to improve the durability of the material without producing any undesirable effects. A number of crosslinking methods have been tested in biological tissues composed mainly of collagen. The aim of this study was to evaluate the in vitro biocompatibility, the mechanical properties, and in vivo calcification of chemically modified bovine pericardium using glutaraldehyde acetals (GAAs) in comparison with glutaraldehyde (GA) treatment. Homsy's tests showed that the most cytotoxic treatment is GA whereas GAA treatments showed lower cytotoxicity. Regarding the mechanical properties of the modified materials, no significant differences in stress at rupture were detected among the different treatments. Zeta-Potential showed higher negative values for GA treatment (-4.9 +/- 0.6 mV) compared with GAA-0.625% (-2.2 +/- 0.5 mV) and GAA-1% (-2.2 +/- 0.4 mV), which presented values similar to native tissue. Similar results were obtained for calcium permeability coefficients which showed the highest values for GA treatment (0.12 +/- 0.02 mm(2)/min), being significantly lower for GAA treatments or non-crosslinked pericardium. These results confirmed the higher propensity of the GA-treated tissues for attraction of calcium cations and were in good agreement with the calcification degree obtained after 60 days implantation into young rats, which was significantly higher for the GA group (22.70 +/- 20.80 mg/g dry tissue) compared with GAA-0.625% and GAA-1% groups (0.49 +/- 0.28 mg/g dry tissue and 3.51 +/- 3.27 mg/g dry tissue, respectively; P < 0.001). In conclusion, GAA treatments can be considered a promising alternative to GA treatment.