99mTc-mebrofenin is used in humans and small animals to assess hepatic function. This study was undertaken to measure hepatic clearance of 99mTc-mebrofenin in healthy horses and to determine whether feed deprivation and increased serum total bilirubin (TBIL) concentration alter 99mTc-mebrofenin clearance. Plasma clearance of 99mTc-mebrofenin was determirned in 7 healthy horses at 0, 48, and 96 hours of feed withholding. Serum TBIL and nonesterified fatty acid (NEFA) concentrations were measured every 24 hours. 99mTc-mebrofenin (4.16 +/- 0.62 mCi, mean +/- SD) was injected into a jugular vein, and blood samples were retrieved from the contralateral jugular vein. A plasma time-activity curve of 99mTc-mebrofenin was generated, from which the area under the curve (AUC) and the T1/2 of the fast-phase (T1/2) and slow-phase (T1/2f) were calculated. Mean +/- SD AUC was 17,700 +/- 4,257, 18,616 +/- 8,078, and 16,168 +/- 6,031 counts per minute (cpm) at 0, 48, and 96 hours, respectively; mean +/- SD T1/2f was 2.80 +/- 0.38 minutes, 3.52 +/- 1.46 minutes, and 3.82 +/- 1.29 minutes at 0, 48, and 96 hours, respectively; median T1/2s was 63.9, 49.2, and 45.8 minutes at 0, 48, and 96 hours, respectively. No difference was detected between the values of AUC, T1/2f, and T1/2s at 0, 48, and 96 hours. There was a significant increase in TBIL with fasting, with a mean +/- SD of 6.3 +/- 1.3 mg/dL at 26 hours. NEFAs increased, reaching a plateau at 48 hours (650 +/- 152 micromol/L). Plasma TBIL concentrations did not correlate with AUC or T1/2s but correlated weakly with T1/2f (r = 0.50). Plasma NEFA concentrations did not correlate with AUC, T1/2s, or T1/2f values. This study suggests that 99mTc-mebrofenin plasma clearance is not affected by feed withholding and that hyperbilirubinemia associated with feed withholding does not affect the hepatic extraction efficiency of this radiopharmaceutical.