Insulin-like growth factor-II (IGF-II) is thought to play a critical role in the development of embryonic tumors such as Wilms' tumor. However, despite highly elevated IGF-II mRNA levels in tumors, IGF-II is not elevated in the serum of patients with Wilms' tumors. Recently high molecular weight forms of IGF-II ('big'- or pro-IGF-II) have been found to be produced by some tumors. In order to prove whether or not high molecular weight forms of IGF-II are produced by Wilms' tumor cells and secreted into the culture medium, we established Wilms' tumor cell lines. After column chromatography of the culture medium, IGF-II and pro-IGF-II concentrations were measured. For pro-IGF-II measurement we established a pro-IGF-II RIA using a rabbit polyclonal antiserum directed against amino acids 7-21 (E7-21) of the E-domain of pro-IGF-II. Gel electrophoresis and Western blotting with anti-IGF-II antibodies revealed a band at 7.5 kDa corresponding to fully processed IGF-II and bands between 10 and 20 kDa. Using pro-IGF-II antiserum, bands between 10 and 25 kDa were detected. We conclude that in vitro cultured Wilms' tumor cells produce and release various forms of 'big IGF-II' with molecular masses between 10 and 25 kDa. It remains uncertain whether these high molecular weight forms of IGF-II represent normal precursors of IGF-II or incorrectly processed IGF-II.