OBJECTIVE To evaluate various biochemical tests as indicators of subclinical liver disease in horses exposed to pyrrolizidine alkaloid toxicosis. METHODS A clinical pathology field study. METHODS Twenty-two clinically normal horses from four properties in the Kimberley region of Western Australia. METHODS Serum samples from each horse were assayed for gamma glutamyltransferase, alkaline phosphatase and aspartate aminotransferase activities, and for serum bile acid concentration, albumin and total protein. Serum protein electrophoresis was performed and their amino acid profiles determined. Bromosulphophthalein half-clearance times were measured. Horses were then subjected to a single liver biopsy. Results were analysed by, variance of group means, the Fisher-Irwin exact test, and by sensitivity and specificity calculation. RESULTS Horses were classified into 2 groups, of 10 unaffected and 12 subclinically affected, on the basis of liver histology. Significant differences between the unaffected and subclinical groups were observed for gamma glutamyltransferase and alkaline phosphatase activities (P < 0.01). Gamma glutamyltransferase had sufficient sensitivity (75%) and specificity (90%) to function as a primary screening test for subclinical liver disease in horses exposed to pyrrolizidine alkaloids. Alkaline phosphatase was useful, but with lower sensitivity (58%). CONCLUSIONS Serum gamma glutamyltransferase activity is a useful screening test for detecting subclinical liver disease in horses exposed to pyrrolizidine alkaloids under field conditions in northern Australia.