The terminal differentiation of the mouse mammary gland epithelium during lactation has been shown to require IGFs and/or superphysiological levels of insulin. It has been suggested that IGF receptor I (IGF-IR), in addition to its well-established role in the mammary gland during puberty and pregnancy, serves as the principal mediator of IGFs at this stage of development. However, our analysis of the expression levels of IGF-IR and the two insulin receptor (IR) splice variants, IR-A and IR-B, has revealed a 3- to 4-fold up-regulation of IR-B transcripts and a 6-fold down-regulation of IGF-IR transcripts and protein during terminal differentiation in the developing mammary gland. IR-B expression was also more than 10-fold up-regulated in murine mammary epithelial cell line HC11 during differentiation in vitro. As already described for the human form, murine IR-B cloned from HC11 exhibited selectivity for insulin as compared with IGFs. When differentiated HC11 cells were stimulated by 10 nm insulin, a concentration that is unable to activate IGF-IR, induction of milk protein and lipid synthetic enzyme gene expression, lactate production, and phosphorylation of Akt were observed. In contrast, on differentiated HC11 cells 10 nm IGF-I or 10 nm IGF-II were able to exert growth-promoting effects only. The lack of response of differentiated cells to low levels of IGFs could not be explained by inactivation of IGFs by IGF binding proteins. Our results suggest a previously unrecognized predominant role for IR-B in the differentiated mammary epithelium.