Insulin-like growth factor-II (IGF-II) is able to interact with three different receptors: the type-I and type-II IGF receptors, and the insulin receptor, although with a lower affinity. This cross reactivity obscures the mechanisms via which the biological activities of IGF-II are mediated. We have synthesized an IGF-II analog, [Leu27]IGF-II, that is highly selective for the type-II IGF receptor. Receptor binding experiments demonstrate a high affinity for the type-II IGF receptor, analogous to synthetic (syn) and recombinant (rec) IGF-II, but no affinity for the type-I IGF and the insulin receptor at concentrations up to 50 and 200 ng/ml, respectively. The lack of affinity for these two receptors is confirmed by biological studies which demonstrated that this analog, in contrast with synIGF-II, did not stimulate [3H]thymidine incorporation in Balb/c 3T3 cells. [Leu27]IGF-II opens new ways to identify which actions of IGF-II are mediated via the type-II receptor and which are due to cross reactivity with the type-I IGF or the insulin receptor.