This study was undertaken to compare various extraction methods for insulin-like growth factor-binding proteins (IGFBPs) from insulin-like growth factor-I (IGF-I) in rat serum systematically, before measurement of IGF-I by radioimmunoassay (RIA). The values obtained in the IGF-I RIA following acid-ethanol (AE), acid-ethanol cryoprecipitation (AEC) and formic acid-acetone (FA) extraction methods were compared with the IGF-I values obtained following high-performance liquid chromatography (HPLC), which was the reference method. Radioligand blots were used to determine the pattern and degree of IGFBP removal by these methods. Over a wide range of circulating IGF-I levels, AE and AEC extraction gave IGF-I levels comparable with those obtained following HPLC. FA extraction resulted in IGF-I levels that were consistently higher (P < 0.01) than those obtained following HPLC and gave non-parallel displacement curves in comparison with recombinant IGF-I standards (P <0.01). Ligand blots demonstrated a similar pattern of IGFBP removal among the three methods with almost complete removal of IGFBP-3 but only 30-40% removal of the lower molecular weight IGFBPs. These lower molecular weight IGFBPs did not interfere with the RIA measurements of IGF-I from AE and AEC extracts. Therefore the AE extraction method of Daughaday, originally validated for use in human serum, is also satisfactory for use in rat serum. The complete removal of IGF-binding activity does not appear essential for accurate measurement of IGF-I by RIA, although this may depend on the specific binding characteristics of the IGF-I antiserum.