We compared two preparations of human insulin-like growth factor (IGF)-I, one isolated from human plasma and the other prepared by recombinant DNA technology, for use in three radioimmunoassays and a radioreceptor assay for IGF-I, two radioreceptor assays for IGF-II, and a protein-binding assay for both IGFs. In the IGF-I assays, iodinated natural IGF-I consistently showed higher binding than did iodinated biosynthetic IGF-I, whether or not the iodopeptides were purified before use by hydrophobic interaction chromatography. Comparing the potency of the unlabeled peptides in the eight assay systems, we found the natural and the biosynthetic IGF-I to be equipotent in every case except for a radioimmunoassay involving a monoclonal IGF-I antibody (the synthetic peptide had 2.4 times greater activity), and an IGF-II radioreceptor assay involving ovine placental membrane receptors (the natural peptide had 10-fold greater activity). We conclude that recombinant DNA-derived IGF-I is suitable for use, both as a standard and as a radioligand, in a wide variety of IGF assays.