Biotinidase is essential for recycling the vitamin biotin and for transferring biotin to proteins, such as histones, suggesting that the enzyme localizes to various cellular and extracellular sites. To better understand the functions of the enzyme, we examined its gene structure and subcellular localization. Using RACE-PCR and a BLAST search, we extended the 5' sequence of the biotinidase gene. Three novel, alternatively spliced variants of biotinidase, 1a, 1b, and 1c, were identified in multiple human tissues. Exon 1c is present only in testes. The sequence of the 5' splice variants, 1a and 1b, suggest that biotinidase localizes to the mitochondria and/or ER, respectively. Using indirect immunofluorescence studies, biotinidase localizes to organelles in the cytoplasm, but not nucleus, of human fibroblasts and Hep G2 cells. Endogenous expression was examined by isopycnic gradient centrifugation of rat liver organelles, which identified an 85kDa biotinidase protein with biotinyl-hydrolase and transferase activities in microsomes and possibly lysosomes. A 48kDa protein, which also reacts with anti-biotinidase, localizes to mitochondria. The 48kDa protein is not N-glycosylated but is biotinylated, is in the inner mitochondrial matrix, but has no biotinyl-hydrolase or transferase activities. The function and validation of the mitochondrial species remains to be determined. The 5' splice variants and organelle fractionation studies indicate that biotinidase is directed to the secretory pathway and perhaps mitochondria.