The tissue distribution and developmental patterns of insulin-like growth factor-II (IGF-II) have not been investigated in rat tissues, primarily because of the lack of an efficient extraction method for IGF-II and a sensitive RIA. IGF-II was extracted from rat tissues by formic acid, and the extract was heated at an acidic pH and treated with acetone. The removal of binding proteins was demonstrated by fast protein liquid chromatography size exclusion column and the elimination of a dilutional bias in the RIA. Using rat IGF-II as standard, we optimized a RIA for the quantification of IGF-II in rat tissues. In adult rats, IGF-II was found in all 15 tissues examined, with the highest concentration in the pituitary, followed by kidney, seminal vesicles, intestine, and serum. This distribution is not only different from that of IGF-I, but also differs from that reported for IGF-II mRNA and IGF-II receptors, suggesting that the rates of synthesis and/or metabolism of IGF-II are tissue dependent. Developmentally, IGF-II levels fell postnatally in most tissues, a pattern similar to that of IGF-II mRNA and IGF-II receptor. This developmental pattern supports the hypothesis that IGF-II is important in early growth and development. A relatively homogeneous distribution was observed in the adult rat brain, a distribution also different from that reported for IGF-II mRNA. In the pituitary, the highest concentration was found in the posterior pituitary, followed by the intermediate and anterior pituitary. In conclusion, IGF-II is found in many tissues of adult rats. This observation supports an autocrine and/or paracrine roles for IGF-II.