Characterization of the local renin-angiotensin system in the rat adrenal zona glomerulosa indicated a dual targeting of renin both to the secretory pathway and mitochondria. To investigate the transport of renin into mitochondria, we constructed a series of amino-terminal deletion variants of preprorenin. One of these variants, lacking the complete signal sequence for the endoplasmic reticulum and 10 amino acids of the profragment, was transported efficiently into isolated mitochondria. The transport was further shown to be dependent on mitochondrial membrane potential and ATP synthesis. Analysis of adrenal RNA revealed the existence of 2 renin transcripts. While one of the transcripts corresponds to the known full-length transcript, the other one lacks exon 1; instead, exon 2 is preceded by a domain of 80 nucleotides originating from intron 1. This domain, as well as the following region of intron 1 being excised, shows all essential sequence elements defining an additional, so-far-unknown exon. The second mRNA possibly derives from an additional transcription start in intron 1 and an alternative splicing process. Translation of this mRNA could result in a truncated prorenin representing a cytosolic form of renin, which is required for transport into mitochondria. This truncated prorenin corresponds exactly to the deletion variant being imported into mitochondria in vitro.