The aim of the present work was to clarify to what extent plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) contribute to the increase in plasma inhibition of tissue-type plasminogen activator (t-PA) observed during pregnancy. It was demonstrated that a monoclonal antibody against PAI-1 almost completely quenched inhibition of single-chain t-PA and most of the inhibition of two-chain t-PA in plasma during the third trimester of pregnancy. The remaining inhibition of two-chain t-PA was to a great extent abolished by a PAI-2 antibody. The second order rate constant (k1) for inhibition of single-chain t-PA by the inhibitor neutralized by the PAI-1 antibody was about 4.8.10(6) M-1.s-1. The conversion of single-chain t-PA to the two-chain form increased the reaction rate with the inhibitor about 3-fold. These kinetic data are comparable with those obtained with PAI-1 in non-pregnancy plasma or with purified PAI-1. From the above results it is concluded that PAI-1 is the primary inhibitor of both single-chain and two-chain t-PA and that PAI-2 is the secondary inhibitor of two-chain t-PA in pregnancy plasma. The concentration of reactive PAI-1 versus gestation age was assayed in plasma from 6 women by binding of PAI-1 to 125I-labelled single-chain t-PA followed by quantitation of the labelled t-PA-PAI-1 complex after separation by SDS-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)