Expression and regulation of the helix-loop-helix (HLH) protein Id in cardiac cells and its negative effect on cardiac gene expression indicate that Id may play a role in cardiac development. To investigate this issue further, we have performed in situ analysis of Id mRNA in developing mouse embryos from Embryonic Days 7 to 15.5 and in neonatal heart. Consistent with its postulated role as an inhibitor of differentiation, Id mRNA was expressed in specific populations of proliferating mesenchymal and epithelial tissues, with its expression declining as differentiation progressed. In addition, patterns of Id mRNA hybridization overlapped considerably with those of two other developmentally important genes, twist and hox 7/7.1, of the HLH and homeobox family, respectively. Id mRNA hybridization was not observed in early epithelial somites or in the myotomal compartment of the somite, suggesting that Id is unlikely to play an inhibitory role in these early myogenic precursors. Id mRNA was expressed in specific regions of the developing nervous system. In the heart, Id mRNA was expressed at high levels in developing cardiac cushions and ridges of the outflow tract and continued to be expressed at high levels in valvular tissue of the neonatal heart.