Analysis of urinary oligosaccharides by thin-layer chromatography (TLC) is used as screening procedure for 10 different lysosomal diseases. We tested the usefulness of HPLC in screening, using a CarboPac PA1 column (Dionex), pulsed amperometric detection (PAD), and post-column derivatization (PCD). Patterns from six types of oligosaccharidoses were compared with normal urinary patterns and with the TLC patterns. PAD appeared to be nonspecific and therefore is applicable only to desalted urine samples. PCD was more specific and applicable to nondesalted urine samples, albeit with a lower resolving power. Peaks in urines from oligosaccharidoses patients were identified on the basis of retention times of commercially available oligosaccharides or TLC bands after isolation and HPLC of the corresponding oligosaccharides. Abnormal oligosaccharide peaks were seen in urines from patients with alpha-mannosidosis, GM1-gangliosidosis (juvenile), GM2-gangliosidosis (Sandhoff disease), Pompe disease, and beta-mannosidosis. HPLC detected no abnormal oligosaccharides in urine from patients with fucosidosis. Although TLC is a simple and reliable screening procedure for detecting classical lysosomal diseases with oligosaccharide excretion, HPLC, by its higher resolution and possibility of quantification, can more generally be used for recognition of abnormal oligosaccharides or detection of increased excretion or content for known oligosaccharides in urine, other body fluids, and cells.