To investigate the behaviour of caffeine (CAF) in patients with Gilbert's Syndrome (GS), a combined oral loading test of caffeine and chenodeoxycholic acid was performed in 14 healthy subjects and 71 patients with GS. Indocyanine green (ICG) kinetics was tested in 50 subjects with GS and in all control subjects. Fasting serum bile acids (SBA) and clearance after CDCA loading were within normal range in normal and GS subjects. No significant difference in levels either of bilirubin or of SBA was observed in GS cases with normal (52 cases, 488 +/- 63 ml/min) or impaired (19 cases, 338 +/- 30 ml/min) caffeine clearance. Eleven GS cases showed altered ICG clearance. No correlation was found between bilirubin and bile acids, CAF or ICG. Fasting SBA were normal even in cases of CAF or ICG altered kinetics, thus excluding structural damage in unconjugated hyperbilirubinemia. CAF altered kinetics in 27% of GS cases may suggest multiple deficits in the hepatocellular metabolism, thus confirming the heterogeneity of this syndrome.