The dimethylmethylene blue (DMB)-based screening procedure for mucopolysaccharidosis and the alcian blue (AB)-based procedure both measure glycosaminoglycan (GAG) content directly in urine. We compared the two procedures. Absorbance per microgram of GAG for DMB was 25 times that obtained with AB, resulting in a requirement for 10 times smaller sample volumes for the former. Recoveries of added heparan sulphate for the AB assay in the absence and presence of added protein (2.5 g/L) were comparable (78-95% and 75-111%), as was the case with the DMB assay. Here, recoveries were generally better (94-103% and 96-100%). The coefficient of correlation (CC) between the two methods measured on 86 reference urine samples was 0.861. For mucopolysaccharidosis urines the CC was higher (0.928). Age-dependent reference values were comparable. Performance of the assays was compared by measurement of GAG content in 24 mucopolysaccharidosis urines. Standard deviation scores (measured value minus mean of reference values divided by standard deviation in reference values) and probabilities for classification in the patient group were calculated and compared. Especially for urines from patients with less distinctly increased GAG content (Sanfilippo, Scheie), scores and probabilities were higher with the DMB assay.