5'-Nucleotidase in the brain of the mouse was examined histochemically. By means of various fixatives, substrate omission, heat effects, pH variation, ionic activators and inhibitors, an antigenic inhibitor (cobra venom antiserum) and substrate specificity, it was shown that the histochemical technique is specific for 5'-nucleotidase, and that it did not depend upon non-specific phosphatases. The enzyme was also show to hydrolyze seven different mononucleotides, but not a dinucleotide, two trinucleotides nor ribose 5-phosphate. Evidence is presented that the hydrolysis of the various mononucleotide substrates may be due either to separate 5'-nucleotidases or to isoenzymes.