This paper deals with a new method for the determination of serum nucleotidase (EC 3.1.3.5). The assay is performed with cytidine-5'-monophosphate as substrate, followed by deamination of the generated cytidine. The principle of the method and the determination of the liberated ammonia by the Berthelot indophenol-reaction are comparable to the Persijn--van der Slik method in which adenosine-5'-monophosphate is used as substrate. The correlation between the results obtained with these two methods was found to be good; the new method has the advantage of higher sensitivity.