The distribution of 5′-nucleotidase in several tissues of rat and mouse has been investigated. The enzyme is greatly specific in dephosphorylating 5-nucleotides. Two 5′-nucleotidases seem to exist: one showing greatest activity at pH 5.0, while the other is most active at pH 7.0-7.5. The localization of these two enzymes is not identical. At the acid pH the deoxyribonucleotides are dephosphorylated faster than the ribonucleotides, while at the neutral pH the ribonucleotides are hydrolysed more rapidly. In contrast to the non-specific phosphatases the nucleotidases can be stimulated by magnesium and manganese ions. The acid nucleotidase can be considered as a lysosomal enzyme. The neutral nucleotidase can be involved in transport processes in capillaries and sinusoids. Other localizations of the enzyme suggest a role of the enzyme in the catabolism of nucleic acids. © 1968 Springer-Verlag.