Helix-loop-helix (HLH) proteins related to the inhibitor of DNA binding/differentiation (Id) serve as general antagonists of cell differentiation. They lack a basic DNA-binding domain and are thought to function in a dominant negative manner by sequestering basic HLH (bHLH) transcription factors that are involved in cell determination and differentiation. Four Id-encoding genes have been shown in mammals, they have a distinct pattern of expression suggesting different functions for each member in different cell lineage. In this study we describe the identification and cloning of two trout cDNAs which encode helix-loop-helix proteins showing a high degree of similarity with mammalian Id family members. One cDNA encodes a trout putative Id1 protein (TId1) that is 63% identical to the human Id1 protein over the entire length and 78% identical within the HLH region. The other cDNA encodes a trout putative Id2 protein (TId2) that shows 82% identity to the human Id2 protein and only one change that is conservative over the HLH region. In the 3' untranslated region. TId2 mRNA exhibits 16 nucleotides upstream from the AATAAA site, a palindromic sequence similar to the cytoplasmic polyadenylation element (CPE) which is also present in Id2 and Id3 mRNAs from mammals and in XIdx/XIdI mRNA from Xenopus. In the fish. TId1 and TId2 are expressed in a tissue-specific manner, with slightly different patterns. During myogenesis. TId1 and TId2 are highly expressed in the myotomal musculature of fish embryos and of early alevins but are down-regulated in that of late alevins. In muscle from juveniles and adults, TId1 and TId2 transcripts are abundant in the slow oxidative fibers while they are absent in the fast glycolytic fibers. This expression pattern suggests that Id genes play a role in the regulation of muscle fiber phenotype in addition to controlling early myogenesis. On the whole, the identification of two HLH-Id encoding genes in a major taxonomic group like teleosts, suggests an early divergence of Id genes in vertebrate evolution. The observation that Id transcripts are present selectively in the slow muscle reveals that their expression is more complicated than previously appreciated.