Recent work from our lab has demonstrated the importance of alternative promoters and variable 5' UTRs in the regulation of two connexin genes. To see whether other connexins also utilize multiple promoters to produce different mRNA isoforms, we screened the mouse EST database for variations in the 5' ends of each connexin EST in UniGene. 5'-RACE analysis of mouse embryo cDNA targeting five candidate genes, Cx31, Cx40, Cx45, Cx46, and Cx47 (approved gene symbols Gjb3, Gja5, Gja7, Gja3, and Gja12, respectively), revealed the existence of multiple previously unknown exons upstream of the coding region that result in variations in the 5' UTR of the mRNA. RT-PCR from 17 different mouse tissues revealed that many isoforms are expressed in a tissue-specific manner, with some being the predominant exons found in the tissues tested. Many of the novel 5' UTRs include upstream open reading frames, suggesting varying translational efficiencies. The expression of alternative 5' UTRs suggests that connexins, like many genes involved in development, require complex regulation at both transcriptional and translational levels.