The 34-kDa translocase of the outer mitochondrial membrane (Tom34) is a putative mammalian-specific factor involved in protein import into mitochondria. We analyzed the genomic sequence of the mouse Tom34 gene and found it has two alternative initial exons. Using reverse transcription and the polymerase chain reaction (RT-PCR), we found that these two mRNAs differs only in the 5'-proximal sequences corresponding to the two initial exons (exon 1a and 1b). Tom34 mRNA with exon 1a (Tom34a) is expressed ubiquitously, while that with exon 1b (Tom34b) is expressed only in mature testicular germ cells. To explore the in vivo function of Tom34 proteins, we generated Tom34-deficient mice by targeted disruption. The Tom34(-/-) mice were viable and grew normally and had a normal Mendelian inheritance pattern. Male as well as female Tom34(-/-) mice were fertile. In vitro-preprotein import into isolated mitochondria showed no apparent difference between Tom34(-/-) and wild-type mice. These results indicate that Tom34 is dispensable for mouse growth and development under optimal conditions.