A new two-site enzyme immunoassay for pepsinogen II(PGII) has been developed. The detection limit of PGII was 0.15 ng/ml (15 pg/tube) and the optimal assay range was 1.5 to 150 ng/ml (150 pg to 15 ng/tube). Mean serum PGII concentrations in normal males and females were 17.2 and 14.6 ng/ml, respectively. The chronological analysis among 5 age groups from 20 to 69 yr, males and females together, revealed that the serum level was low (11.9 ng/ml) between 20-39 yr, gradually increased from 40 yr of age and attained the maximum (22.5 ng/ml) in the 50-yr group. Total gastrectomy markedly lowered the serum level to 1.9 ng/ml, suggesting that the majority of PGII in serum was derived from the gastric mucosa. This assay makes it possible to measure trace amounts of PGII derived from extragastric as well as gastroduodenal tissues.