The objective of this study was to determine the effect of high pressure homogenisation (HPH) on alkaline phosphatase activity and plasmin and plasminogen-derived activities in raw whole bovine milk. Milk (approximately 4% fat) was treated by two-stage conventional homogenisation (18 MPa) or single or two-stage HPH at 50, 100, 150 or 200 MPa. Inactivation of plasmin and plasminogen-derived activities was evident in conventionally homogenised samples, and increased as HPH pressure increased. Two-stage HPH reduced both activities to a greater extent than single-stage HPH. Milk inlet temperature had a significant effect on residual plasmin and plasminogen activities of HPH-treated milk samples, especially those treated at 50 MPa. Inactivation of plasmin and plasminogen on HPH-treatment (150 MPa) of milk samples of varying fat contents (0–10%) was also investigated; there was a curvilinear relationship between residual plasmin and plasminogen-derived activities and fat content in the ranges 0–2% and 0–4%, respectively, with little additional inactivation at higher fat contents. Thus, indigenous proteolytic activity of milk is clearly affected by HPH. However, all homogenised milk samples retained active alkaline phosphatase, indicating that thermal conditions during HPH did not equate to that of conventional high temperature short time pasteurisation, and that the wide range of forces experienced by milk during HPH treatment does not inactivate the latter enzyme.