Measuring the content of immunoreactive pancreatic phospholipase A2 (PLA2; EC 3.1.1.4) and the catalytic activity of PLA2 in serum samples from five patients with acute pancreatitis, we found no correlation between these two measurements overall. To test the specificity of the method for catalytic PLA2, we measured PLA2 activity in serum samples before and after immunoadsorption with an antiserum to human pancreatic PLA2. The results suggest the presence of at least two immunologically distinct PLA2 enzyme proteins in sera from these patients. One of the enzymes is pancreatic in origin and may exist in active, inactive, or inhibited form. The activity profile of the second PLA2 enzyme in serum during acute pancreatitis differs from that for other common pancreatic enzymes. In the present experiment, the catalytic activity was not removed by treatment with the anti-human pancreatic PLA2 antiserum. The source of this second PLA2 activity is unknown. Some samples contained increased activities of both PLA2 forms.