The most widely used radioisotope in autoradiography is tritium (³H). Tritium allows a resolution of about 1 micrometer with the light microscope and close to 0.1 micrometer with the electron microscope.

Since hydrogen is common in biological molecules, a wide-range of ³H-labeled compounds are potentially available for use in autoradiography. ³H-amino acids are used for locating newly synthesized proteins, ³H-thymidine is used to monitor DNA synthesis, ribonucleiodies such as ³H-uridine or ³H-cytidine are used to localize newly made RNA molecules, and ³H-glucose is used to study the synthesis of polysaccharides.