Alkaline phosphatase (ALP) is an enzyme present in intestinal mucosa, bile, bone, and renal tubule cells. We sought to assess the diagnostic and prognostic relationships of total ALP (ALPt) activity and that of intestine-derived ALP (ALPi) in serum and peritoneal fluid of 126 horses with colic. ALPt and ALPi activities were measured in both serum and peritoneal fluid by using both standard and L-phenylalanine-based buffers, respectively. Neither ALPt nor ALPi activity were useful in classifying type or severity of intestinal damage. ALPt and ALPi activities in peritoneal fluid were lowest in horses suffering from simple medical colic (39 international units [U]/L [19–60 U/L]; versus 31 U/L [16–44 U/L], median [interquartile range], P < .001) and nonstrangulated surgical lesions (45 U/L [30–62 U/L] versus 36 U/L [23–54 U/L], P < .001), and highest in surgical cases with suspected ulceration (109 U/L [60-1,113 U/L] versus 83 U/L [52–970 U/L], P < .001), strangulation (114 U/L [69–240 U/L] versus 94 U/L [56–191 U/L], P < .001), peritonitis (313 U/L [110-2,227 U/L] versus 283 U/L [91-1,800 U/L], P < .001) or intestinal rupture (687 U/ L [205–852 U/L] versus 564 U/L [166–732 U/L], P < .001). Higher ALPt and ALPi activities in peritoneal fluid were associated with greater intestinal damage, increased probability of surgery, and a worse prognosis. The use of L-phenylalanine buffer in both serum and peritoneal fluid did not improve the sensitivity of the test. Based on these results, total ALP activity in peritoneal fluid may help in identifying ischemic or inflammatory bowel lesions in horses with acute colic.