Using the Ektachem-700 multilayer film analyzer, we defined age- and sex-specific reference intervals for 20 analytes in sera from a healthy population of neonates and children ages one to 19 years. Upper and lower normal reference intervals for each analyte were determined by nonparametric methods as the 0.975 and 0.025 fractiles, respectively. Newborns have lower concentrations of total protein and albumin, and higher concentrations of phosphate, bilirubin, and enzymes in serum than older children do. Concentrations of urea, glucose, calcium, phosphate, and bilirubin change rapidly postnatally. Outside the neonatal period, no significant age- or sex-related difference was found for plasma glucose, serum amylase, conjugated or unconjugated bilirubin, or lipase. There was no sex-related difference in reference intervals for albumin, total protein, calcium, phosphate, or urea. However, concentrations of uric acid and creatine kinase are much higher in postpubertal boys than in girls. Alkaline phosphatase values peak later in boys. Except for lactate dehydrogenase and gamma-glutamyltransferase, the reference intervals defined here do not differ strikingly from data derived with use of other analyzers. The age- and sex-related trends are independent of method. However, each laboratory should determine the degree to which these reference ranges can be directly applied to analyses performed with another analyzer.