Six pairs of identical twin cows during late lactation (213 d) were used to study the effect of milking frequency (twice vs. once daily) and bST during once daily milking on the activity of plasminogen activator, plasminogen, and plasmin in milk. Less frequent milking increased the activity of plasminogen, plasmin, and plasminogen activator in milk. The ratio of plasminogen to plasmin, a measure that is independent of milk volume, decreased during less frequent milking, suggesting that at least part of the increase in activity of plasmin was due to the accelerated conversion of plasminogen to plasmin. Changes in the activity of plasminogen and plasmin in milk were positively correlated with increases in the concentrations of milk BSA and plasma lactose, both of which are indicators of disruption of tight junctions between mammary epithelial cells, indicating that paracellular leakage may have contributed to increased protease activity in milk during less frequent milking. No correlation existed between changes in plasminogen activator and indicators of tight junction disruption, suggesting that increased activity of plasminogen activator in milk was not due to leakage across the mammary epithelium, but rather to increased local production in the mammary gland. Administration of bST during once daily milking did not significantly affect milk protease activity.