With the aim to produce insulin-like growth factors (IGF) with enhanced specificity for the type 1 or type 2 IGF receptors, three mutants of IGF II have been prepared and expressed in NIH-3T3 cells. IGF II mutated at Tyr27 to Leu and Glu showed a 25- and 54-fold decrease in affinity for the type 1 IGF receptor and a 3.4- and 9.2-fold decrease in affinity for the type 2 IGF receptor. IGF II mutated at Phe48 to Glu showed a 18-fold decrease in affinity for the type 2 IGF receptor and a 2.8-fold decrease in affinity for the type 1 IGF receptor. These affinities were measured in radioreceptor assays using type 1 or 2 IGF receptor overexpressing cells. Data obtained on receptor cross-linking and thymidine incorporation assays confirmed the results of the radioreceptor assays. It is concluded that mutations of Tyr27 preferentially decrease binding to the type 1 IGF receptor and of Phe48 to the type 2 IGF receptor, either by the loss of a residue involved in receptor binding or by preferentially destabilizing the region involved in receptor binding.