Choline dehydrogenase (CHDH, EC 1.1.99.1) was purified from rat liver mitochondria, and the amino terminal sequence was determined and used to clone a full-length cDNA encoding a protein precursor (CHDHp) of 599 amino acids (64kDa). Sequence analysis identified a possible processing site that meets the requirements of IMP in comparison to the previously determined N-terminal sequence of mature rat CHDH. This suggested that the precursor might be processed in the intermembrane space. Confocal imaging showed that expression of the CHDHp-GFP fusion gene in NIH-3T3 cells led to fusion proteins being targeted to mitochondria. In addition, expression of a recombinant version of the CHDHp gene in Saccharomyces cerevisiae led to enrichment of the target protein in the mitochondrial inner membrane. The expressed protein conferred choline dehydrogenase activity, suggesting that both functional domains (FAD and the iron sulfur cluster) were properly assembled and that the mature CHDH was appropriately located in the inner mitochondria membrane.