Unlike most proteases, tissue-type plasminogen activator (t-PA) is secreted from cells as an active, single chain "proenzyme" whose catalytic efficiency is comparable with that of the corresponding mature, two-chain enzyme. We have previously suggested that the absence of the "zymogen triad" (Asp194-His40-Ser32; chymotrypsin numbering) contributes to this unusually high enzymatic activity of single chain t-PA. Consistent with this prediction, the single chain form of a variant of t-PA containing the zymogen triad displayed dramatically reduced activity toward synthetic substrates. Activation cleavage of this variant, however, resulted in a mature, two-chain enzyme with full catalytic activity. To further examine the functional significance of the zymogen triad, we used site-specific mutagenesis to construct a variant of t-PA, t-PA/R275E,A292S,F305H, that contained this triad but could not be converted into its two-chain form by plasmin. Characterization of this variant demonstrated that the presence of the zymogen triad specifically suppressed plasminogen activation by single chain t-PA in the absence of fibrin. In addition, these studies indicated that, like wild type t-PA, zymogen activation of this variant could be accomplished by binding to the co-factor fibrin. The combination of full activity in the presence of fibrin and reduced activity in its absence resulted in novel variants of t-PA that displayed dramatically enhanced stimulation by fibrin. While the presence of fibrin increased the catalytic efficiency of t-PA toward plasminogen by a factor of approximately 520, this stimulation factor increased to 130,000 for t-PA/R275E,A292S,F305H. Plasmin-resistant, zymogen-like variants of t-PA, therefore, may represent thrombolytic enzymes with enhanced "clot selectivity."