Background: Jaundice is one of the most common and one of the vexing problems that can occur in newborns. A newborn screening test for biotinidase deficiency has been added to many national screening programmes. Aim: To clarify the problem of false-positive screening tests in neonates, especially in term babies, we evaluated the biotinidase activity in the serum of fullterm, premature and small-for-dates newborn infants with jaundice. Methods: 1296 fullterms (controls N=426), 246 prematures (controls N=86) and 156 small-for-dates babies (controls N=38) aged 2–3 days with jaundice were included in the study. In jaundiced neonates and controls, 3.0 ml of blood was drawn for the evaluation of total bilirubin (t.bil), liver enzymes and biotinidase activity in the serum using a fluorimetric method. In order to test whether or not t.bil causes an artifact in the previous method, biotinidase activity was also evaluated in a number of jaundiced newborns using an HPLC method. Additionally, a preliminary in vitro experiment was carried out to test whether t.bil is an inhibitor of the enzyme. Results: Biotinidase activities in the group of controls of prematures (3.30±1.2 mmol/min/l) and small-for-dates babies (3.34±0.8 mmol/min/l) were lower than those of term babies (4.99±1.1 mmol/min/l, p<0.001). T.bil and liver enzymes showed a statistically significant inverse correlation with biotinidase activity (p<0.001) in all the jaundiced infants of this study. Additionally, biotinidase activity, evaluated in a number of neonates with both fluorimetric and HPLC methods showed similar results. Preincubation of the serum enzyme with t.bil (>10 mg/dl) resulted in a 50% or more inhibition. Conclusions: (a) Low biotinidase activity was found in term babies, prematures and small-for-dates with jaundice. (b) The low activity of the enzyme could be due to their impaired liver function. (c) The high t.bil levels in the studied groups may play the role of an “inhibitor” of the enzyme. (d) Gestational age as well as t.bil levels should always be written on Guthrie cards for a correct evaluation of biotinidase activity.