A number of tightly regulated proteolytic enzyme systems, including the plasminogen activation cascade and matrix metalloproteases, play integral roles in the remodelling of extracellular matrices during pregnancy and parturition. This study assessed these labour-associated changes in protease activity in human gestational tissues. Amnion, choriodecidua and placenta collected from women before (at caesarean section, not in labour), during (at caesarean section, in labour) and after (spontaneous-onset labour, normal vaginal delivery) labour were examined on gelatin-substrate SDS-PAGE zymography. All tissues displayed major 55 kDa plasminogen-dependent activity that was abolished by the serine protease inhibitors (10 mmol phenylmethyl-sulphonylfluoride l-1, 100 mmol epsilon aminocaproic acid l-1, 1 mmol Glu-Gly-Arg chloromethylketone l-1). The enzymic activity was identified as urokinase plasminogen activator on the basis of its co-migration with reference standard and western blot analysis, and did not vary with labour status. An additional protease with an apparent molecular mass of approximately 90 kDa was detected in all tissues. Densitometric measurement of these tissues showed a significant (P < 0.05) increase in this enzyme activity with labour onset. Heavy metal chelators (1 mmol 1.10 phenanthroline l-1 and 10 mmol EDTA l-1) selectively blocked the 90 kDa activity, consistent with the proposal that it is a metalloprotease. Co-migration with reference standard and western blot analysis confirmed the identity of this protease as the matrix metalloprotease 9 (MMP-9). Immunoreactive MMP-9 protein was also significantly (P < 0.05) increased during and after labour compared with before labour in all tissues examined. It is proposed that the upregulated expression of MMP-9 is involved in fetal membrane rupture and placental separation during and after labour onset, respectively. In conclusion, the regulated repertoire of protease activities expressed by human gestational tissues implies an important role for matrix-degrading enzymes during human parturition.