The process of posttranslational modifications of IGF-II likely has important physiological consequences. In addition to mature IGF-II, glycosylated proIGF-II (156-amino acid peptide) and two glycosylated "big" IGF-II forms, IGF-II(1-104) and IGF-II(1-87), have been identified in the human circulation. Due to lack of an appropriate methodology, different IGF-II isoforms have not been demonstrated and characterized in the rat circulation, thus preventing a better understanding of the physiological and pathological roles of IGF-II. In the present study, we have characterized each IGF-II form and assessed its content in the rat circulation throughout life time by using a highly sensitive Western blot analysis, which is void of the IGFBP interference and distinguished all IGF-II forms. For the first time, we demonstrated the presence of IGF-II variants, including proIGF-II, IGF-II(1-87) and mature IGF-II, in the rat circulation during postnatal life, challenging the current impression that IGF-II is absent from sera of adult rats. ProIGF-II is glycosylated and is the predominant form in the rat circulation. Endoproteolytic processing of proIGF-II was clearly activated in fetal, neonatal and pregnant rats, likely reflecting its involvement in fetal development through the generation of specific forms of IGF-II (e.g. mature IGF-II) that are required for their distinct biological functions. Taken together, our data also suggests that serum IGF-II profiles may reflect underlying physiological conditions.