We have shown that fibrin-based small-diameter tissue-engineered blood vessels (TEVs) exhibited considerable mechanical strength and could withstand implantation in the jugular veins of lambs, where they remained patent for 15 weeks. The microtopology of fibrin matrix is influenced by the concentration of fibrinogen and calcium, whereas fibrinolysis and matrix remodeling are affected by the presence of the fibrinolytic inhibitor aprotinin. Here we report the effects of these components on two key properties of TEVs, namely mechanical strength and vasoreactivity. We found that high concentrations of fibrinogen or calcium decreased significantly both strength and reactivity. Surprisingly, aprotinin increased mechanical strength but decreased vascular reactivity in a dose-dependent manner. Transforming growth factor beta(1) (TGF-beta(1)) and insulin had a moderate effect on mechanical strength but significantly enhanced reactivity, through receptor- and non-receptor- mediated pathways. In addition, the combination of TGF-beta(1), insulin, and aprotinin resulted in significant improvement of both properties. Our data suggest that the microtopology of fibrin matrix and the rates of fibrinolysis and extracellular matrix synthesis may affect the properties of TEVs significantly. They also indicate that biomaterial and culture parameters may have differential effects on mechanical properties versus vascular reactivity and, therefore, engineering blood vessels under conditions that maximize tissue strength may not always result in optimal function. Instead, strength and reactivity must be used in concert for more accurate evaluation of tissue-engineered vascular constructs.