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.