Six variants of tissue-type plasminogen activator (t-PA) were produced in mouse C127 cells using a bovine papilloma virus expression vector. All variants lacked the growth factor (G) domain and the first kringle domain (K1) and three of the variants also lacked the finger domain (F). Furthermore, the specific changes, Lys 277 → Val and Asn 448 → Gln were introduced into some of the molecules. The variants were denoted K2P, K2P(Val277), K2P(Gln448), FK2P, FK2P(Va1277), and FK2P(Gln448). Amino acid sequence analyses revealed that the variants were proteolytically processed in the amino terminus and at Arg 275-Ile 276 in the same way as the full sized molecule. A proteolytically sensitive site was identified in the F domain at Arg 27-Ser 28. The two variants that lacked glycosylation at Asn 448, K2P(GIn448) and FK2P(GIn448), were cleaved at Arg 449-Thr 450, indicating that the oligosaccharide normally present at Asn 448 protects this site against proteolysis. The fibrin affinity for all variants was markedly reduced compared with normal t-PA. The plasminogen activator activity of all variants was stimulated by cyanogen bromide fragments of fibrinogen. In an indirect chromogenic assay K2P and K2P(Val277) showed specific activities that were 23% and 36%, respectively, of that of wild type t-PA, while the corresponding non-glycosylated variant K2P(Gln448) was as active as t-PA. The activity of the three F domain-containing variants were between 88 and 98% of the value determined for t-PA. When the specific activity was determined with the fibrin plate assay all variants were found to have higher specific activities than t-PA (1.8–4.7 fold). The lack of correlation between the activity of t-PA and the variants in these two assays indicate that the reaction mechanism may differ between the variants and wild type t-PA. The kinetic constants K m and k cat were determined for two-chain forms of t-PA and the variants with the chromogenic peptide substrate DIleProArgpNA. The results show that the t-PA heavy chain is not affecting the reaction with small peptide substrates as the K m and k cat values were essentially identical for t-PA, K2P, and FK2P (K m, 0.18–0.21 mM and k cat, 8.8–11.1s −1). The values for the two non-glycosylated variants K2P(Gln448) and FK2P(Gln448) were 0.28mM, 9.4s −1 and 0.24mM, 5.3s −1, respectively. Interestingly, the Val277 variants showed significantly reduced K m values, suggesting that Lys 277 is important for the substrate interaction. For the variant K2P(Val277) K m and k cat were 0.06mM and 6.5s −1, respectively, and for FK2P(Val277) the corresponding values were 0.06mM and 6.0s −1.