Total somatomedins from milk of bovine somatotropin-treated cows were isolated and characterized to determine the relative amount of the three amino acid N-terminally truncated form of IGF-I (destripeptide IGF-I). The somatomedin fraction was isolated using organic solvent and solid-phase extractions followed by preparative reverse phase HPLC and affinity chromatography. The overall yield of IGF-I was 28%, and destripeptide IGF-I was recovered with similar efficiency. The isolated somatomedins were resolved by capillary zonal electrophoresis and identified using recombinant somatomedin standards. The concentration of destripeptide IGF-I relative to full length IGF-I was determined by amino terminal sequencing and by bioassay. Results from these experiments indicated that the level of destripeptide IGF-I in milk from somatotropin-treated cows was less than 3% of the IGF-I concentration. Destripeptide IGF-I is therefore a minor component of the somatomedins present in milk from treated cows and does not contribute significantly to the proliferative activity of this milk. (Pediatr Res 32: 296–300, 1992)