Summary A recombinant low M r derivative of single chain urokinase-type plasminogen activator consisting of amino acids Leu 144 through Leu 411 (rscu-PA-32k) was conjugated to F(ab′) 2 -fragments of a murine monoclonal antibody (MA-15C5), directed against fragment D-dimer of crosslinked human fibrin, by means of the crosslinking reagent N-succinimidyl 3-(2-pyridyldithio) propionate (rscu-PA/MA-15C5-F(ab′) 2 ). The conjugate was purified by immunoadsorption on a monoclonal antibody directed against urokinase followd by affinity chromatography on insolubilised fibrin fragment D-dimer. Recoveries were 26±3.1% for rscu-PA-32k and 27±2.7% for total protein (mean±SD, n=3). The purified conjugate contained 1.0±0.03 mol of F(ab′) 2 -fragment per mol of rscu-PA-32k. The specific amidolytic activity on a chromogenic substrate for urokinase was 2 and rscu-PA-32k, whereas the specific fibrinolytic activity on fibrin plates was 29000 iu/mg u-PA antigen in the conjugate as compared to 53000 iu/mg u-PA antigen in rscu-PA-32k. Activation of plasminogen by rscu-PA-32k/MA-15C5-F(ab′) 2 obeyed Michaelis-Menten kinetics with a K m value of 0.71±0.26 μM and a k 2 value of 0.00023±0.00010s −1 (n=3), as compared to K m =1.6 μM and k 2 =0.00028s −1 for rscu-PA-32k. Both rscu-PA-32k/MA-15C5-F(ab′) 2 and rscu-PA-32k induced dose- and time-dependent lysis of a 125 I-fibrin labelled human plasma clot immersed in citrated human plasma. Fifty per cent lysis in 2h was obtained with a concentration of 6.4 μg/ml (n=4) for rscu-PA-32k, whereas only 1.2μg u-PA/ml (n=4) was required of rscu-PA-32k/MA-15C5-F(ab′) 2 . We conclude that low M r scu-PA can be targetted to a fibrin clot with the F(ab′) 2 -fragment of a fibrin-specific antibody, resulting in a 5-fold increased thrombolytic potency, which is comparable to that of intact scu-PA coupled to intact antibody. These fragments may serve as preferred moieties for the construction of recombinant chimaeric scu-PA/antibody proteins for fibrin-targetted thrombolytic therapy.