Recent advances in autoradiographic technique make it possible to localize quite a diverse class of soluble and/or diffusible substances. However since the molecules in question could move or be displaced at any time during preparation it is necessary to employ chemical or physiological tests to prove the validity of the localization. For two classes of compounds this can be done quite readily. 3H-ouabain, a cardiac glycoside that binds specifically to the Na pump, can be monitored for its effect on Na transport and the binding itself can be controlled by manipulating specific ligands known to modulate the ouabain-Na pump interaction. Acetylcholine can be readily analyzed by chemical means and its solubility characteristics allow one to extract it even from osmium-fixed embedded sections. Thus it is possible to monitor for the presence of acetylcholine in tissue throughout the processing for autoradiography and even chemically characterize small areas of tissue by microdissection of sectioned material.