Hydrothermal isometric tension (HIT) testing and high-performance liquid chromatography were used to assess the molecular stability and cross-link population of collagen in the four valves of the adult bovine heart. Untreated and NaBH(4)-treated tissues under isometric tension were heated in a water bath to a 90 degrees C isotherm that was sustained for 5 h. The denaturation temperature (T(d)), associated with hydrogen bond rupture and molecular stability, and the half-time of load decay (t(1/2)), associated with peptide bond hydrolysis and intermolecular cross-linking, were calculated from acquired load/temperature/time data. An unpaired group of samples of the same population was biochemically assayed for the types and quantities of enzymatic cross-links present. Tissues known to endure higher in vivo transvalvular pressures had lower T(d) values, suggesting that molecular stability is inversely related to in vivo loading. The treated inflow valves (mitral and tricuspid) had significantly lower t(1/2) values than did treated outflow valves (aortic and pulmonary), suggesting lower overall cross-linking in the inflow valves. Inflow valves were also found to fail during HIT testing significantly more often than outflow valves, also suggestive of a decreased cross-link population. Inflow valves may be remodeling at a faster rate and may be at an earlier state of molecular "maturity" than outflow valves. At the molecular level, the thermal stability of collagen is associated with in vivo loading and may be influenced by the mature, aldimine-derived cross-link, histidinohydroxylysinonorleucine. We conclude that the valves of the heart utilize differing, location-specific strategies to resist biomechanical fatigue loading.