Mutations in the gene MID1 are responsible for the X-linked form of Opitz syndrome, a genetic disorder that primarily affects the development of midline structures. Several mRNA isoforms with variant 5' ends have been reported for MID1, suggesting the presence of alternative transcription initiation sites. However, the genomic organization and expression pattern of the heterogeneous MID1 5'-untranslated regions (UTRs), as well as the promoter regions regulating their transcription, have not been determined. We now report the characterization of alternative MID1 first exons and their associated promoters in human and other species. Multiple transcription initiation sites, dispersed over 250 kb on Xp22, were found to give rise to five MID1 isoforms in human that differed in their 5'-UTRs, but contained identical coding sequence. These alternative MID1 transcripts showed distinct expression patterns with the most 5' first exon being adipose-specific and another exon being placenta-selective, whereas other 5'-UTRs seemed to be ubiquitous. Consistent with the expression data, the putative promoter regions upstream of the variant first exons also varied in their strength and tissue specificity in transfection experiments. Similar to the human gene, heterogeneous Mid1 isoforms were also identified in mouse and rat by 5'-RACE, which revealed that three of the five alternative first exons isolated in human are also used in rodent. Comparison of MID1 promoter regions from several mammals indicated a high level of identity suggesting evolutionary conservation. Together, these results suggest that alternative promoters are important in the complex transcriptional regulation of MID1 expression in several species.