Reversed-phase chromatography (RPC) was used to resolve two variants of recombinant amino terminal methionyl residue (N-Met) insulin-like growth factor-I (IGF-I) with the same amino acid constitution but different disulphide linkages. Following radioiodination, equilibration with plasma and size exclusion chromatography at neutral pH the major form on RPC (approximate abundance 60%) demonstrated greater than 80% binding to 150 kDa and 40-50 kDa IGF binding proteins. This peptide has the RPC elution characteristics and disulphide assignment (Cys6-Cys48, Cys18-Cys61, Cys47-Cys52) of authentic with mismatched disulphides (Cys6-Cys47, Cys18-Cys61, Cys48-Cys52; N-Met IGF-I peak 1 peptide) demonstrated less than 15% binding under similar conditions. Potency of the peptides was investigated in competitive IGF-I plasma binding protein and L6 myoblast radioreceptor assays. The peak 2 peptide proved equipotent to purified ovine plasma IGF-I in each system but by contrast the peak 1 peptide was 40-fold and 200-fold less potent in the binding protein and radioreceptor assays respectively. Biological potency was examined in a non-competitive assay based on incorporation of [3H]leucine into confluent cultures of L6 myoblasts. In this system the N-Met IGF-I peak 1 peptide proved 15-fold less potent than the peak 2 peptide with correct disulphide linkages. Refolding variants may prove useful in establishing structure/function relationships for IGF-I.