The whole molecule of human somatomedin C (SM-C) prepared by the total synthesis method was used as an antigen to produce an antiserum for a radioimmunoassay. Since plasma proteins that bind SM-C interfere with the assay, a method was developed that uses acid dissociation followed by C-2 cartridge extraction to strip SM-C from its binding proteins before assay. This assay has no cross-reactivity with human proinsulin or insulin-like growth factor II (IGF-II). The SM-C values in 339 normal subjects showed age-dependence, increasing from childhood to a peak at age 14 to 16 years and decreasing sharply before adulthood. In adults, the SM-C values decreased gradually with age. All 13 patients with acromegaly who were tested had an increased SM-C value, with no overlap with the normal range. The 12 patients with prolactinoma but non-growth-hormone-producing pituitary tumor had no increase in SM-C. Two children with pituitary deficiency had low SM-C values; one of these children received growth hormone therapy, and his SM-C value increased from undetectable to normal. By three weeks after discontinuation of the therapy, his SM-C value was again undetectable. Of 20 children with short stature and constitutional delay of growth and development, SM-C was below normal in 70 percent and normal in 30 percent. Two patients with malnutrition had below-normal SM-C values.