The murine dominant negative helix-loop-helix (dnHLH) proteins inhibit the activities of bHLH transcription factors in diverse cell lineages (Benezra et al. [1990] Cell 61:49-59; Christy et al [1991] Proc. Natl. Acad. Sci. U.S.A. 88:1815-1819; Sun et al [1991] Mol. Cell Biol. 11: 5603-5611; Riechmann et al. [1994] Nucleic Acids Res. 22:749-755). Currently, there are four members in the dnHLH family, Id1, Id2, Id3, and Id4. In this report, we have performed a detailed comparative in situ hybridization analysis to examine their expression pattern during post-gastrulational mouse development. Id1, 2, and 3 are expressed in multiple tissues, whereas Id4 expression can only be detected in neuronal tissues and in the ventral portion of the epithelium of the developing stomach. The regions where Id1-3 genes are expressed, such as gut, lung, kidney, tooth, whisker, and several glandular structures, are undergoing active morphogenetic activities. The expression patterns of Id1, 2, and 3 overlap in many organs, except in the tissue derived from primitive gut. In the latter, Id1 and Id3 signals are detected in the mesenchyme surrounding the epithelium, whereas Id2 is expressed within the epithelium. The difference in the patterns of expressions of Id2-3 and Id4 suggest that the dominant negative transcriptional activity of these two subclasses of the Id family may have different physiological consequences.