The pH optimum of 5-nucleotidase from human thyroid, liver and aorta has been studied. Serum 5NT from patients with hepatobiliary obstruction has also been studied. Methods for the partial purification of thyroid 5NT and for the separation of alkaline phosphatase and 5NT in liver and serum are presented. The pH optimum for human 5NT is shown to be about 6.8 and it is suggested that in the light of these results, methods for the measurement of serum 5NT should be reexamined.