To localize the binding region of porcine tissue-type plasminogen activator (EC 3.4.21.31) (t-plasminogen activator) to heparin, functionally active A and B chains (molecular mass of each 33 kDa) were separated from the two-chain t-plasminogen activator after mild reduction and alkylation. The A chain bound to fibrin-Sepharose, but not to heparin-Sepharose. In contrast, the B chain showed amidase activity toward HD-Ile-Pro-Arg-p-nitroanilide (S-2288) and a high affinity for heparin-Sepharose, but no affinity for fibrin-Sepharose. Plasminogen activator activity of the B chain was stimulated by heparin (about 3-fold), but not by fibrin. On the other hand, the elastase digestion fragments of plasminogen, kringle 1-3 and kringle 4, had no affinity for a heparin-Sepharose column, whereas the other fragment, Val442-plasminogen, efficiently bound to the column and was eluted with 1.6 M KSCN-containing buffer. The stimulatory effect of fibrin on two-chain t-plasminogen activator-catalyzed Val442-plasminogen activation was clearly diminished by heparin. These results suggest that heparin can form a complex with both t-plasminogen activator and plasminogen molecules through their catalytic regions located in each B chain, and that the heparin connection between t-plasminogen activator and plasminogen may improve the plasminogen activation kinetics by making a situation in which t-plasminogen activator is easily approachable to plasminogen.