Id-1 is one of the four related helix-loop-helix Id proteins that act as inhibitors of the basic helix-loop-helix (bHLH) transcription factors that control cell differentiation, development and carcinogenesis. We previously found that Id-1 regulated the growth, differentiation, apoptosis and invasion of mouse mammary epithelial cells in culture. Using the techniques of immunohistochemistry and in situ hybridization, we now show that Id-1 gene expression is specifically detected in the epithelial cells of growing ductal structures during early pregnancy. Using adjacent sections, we determined that Id-1 was expressed in keratin 8/18 positive cells. We also demonstrated that the up-regulation of Id-2 during late pregnancy correlated with the down-regulation of Id-1. Using the yeast-two hybrid system, we identified the bHLH transcription factors, ITF-2A and ITF-2B, as the Id-interacting proteins. The levels of expression of these two splice variants did not change during the transition from growing ductal structures to differentiated alveoli. Therefore Id-1 and Id-2, but not the ubiquitous bHLH proteins, appear to represent the key factors whose expression is modulated during different stages of pregnancy in mouse mammary glands. (c) 2008 Elsevier Inc. All rights reserved.