Concentrations of 34 biochemical constituents of sera were determined on 998 randomly selected urban school children and adolescents aged 8–18 years from Zagreb, Croatia. Reference intervals were obtained by using non-parametric methods to estimate 2.5 and 97.5 percentiles of distribution as upper and lower normal reference intervals, according to the IFCC recommendations. These were compared to reference intervals in the healthy adult population, aged 20–30 years from the same geographical area. Serum glucose, potassium, sodium, chloride, magnesium, iron, zinc, total serum proteins and electrophoretic fractions, and amylase, did not show age or sex differences; total serum bilirubin, total calcium, phosphate, high density lipoprotein cholesterol, total iron binding capacity, unsaturated iron binding capacity, copper, aspartate aminotransferase, alkaline phosphatase, cholinesterase, creatine kinase, and lactate dehydrogenase had higher reference intervals than the adult population. Urea, creatinine, urate, alanine aminotransferase, γ-glutamyltransferase, total cholesterol and low density lipoprotein-cholesterol, and triglycerides had lower reference intervals than the adult population.