The optimal conditions for the cytochemical localization of 5'-nucleotidase (AMPase) in the mouse lymphocyte have been established. Quantitative monitoring of the effects of fixation and the components of the cytochemical medium showed that the cytochemistry can be performed under conditions that do not lead to loss of AMPase activity, and also under conditions where penetration of the substrate into the cell has occurred. The cytochemical reaction product was seen only on the surface of a proportion of splenic lymphocytes, regardless of the fixative used. Biochemical data confirmed that AMPase is an ectoenzyme and is the only protein in splenic lymphocytes capable of catalysing the hydrolysis of AMP. The activity of 5'-nucleotidase was studied also by harvesting cells either from thymus or spleen of A/ST or Cd-1 mouse strains. The enzymatic activity in splenic lymphocytes was more than six time higher than the activity of intact thymus cells. Cytochemically it was evident that within splenic lymphocytes there was a distinct population of lymphocytes with readily demonstrable AMPase activity, and another with no cytochemically demonstrable AMPase activity. It was concluded that murine lymphocytes vary in their activity of AMPase, and that the enzyme is exclusively confined to the cell surface.