5′‐nucleotide phosphodiesterase (5′‐NP) isoenzymes were separated from the serum of human primary liver cancer (PLC) patients by 4–20% tubular polyacrylamide gel gradient electrophoresis. Electrophoresis showed II isoenzymes in which bands I, V and VI (from anode to cathode) were all absent in 125 normal control sera; bands V and VI were detectable in 31(73.8%) of 42 patients with PLC, but absent in 70 cases of other diseases. Bands V and VI may represent specific isoenzyme bands of PLC, comprising a new addition to the markers used in the diagnosis of this disease. By the same method, it was also found that the abnormal serum isoenzyme bands of PLC correspond to those found in the cord blood sera of the newborn and fetus. These abnormal isoenzymes diminish gradually after birth and all disappear when children reach the age of 10.