The 2.3 kb mRNA that codes for Cytochrome P-450c27 (CYP27) has an unexpectedly long 5’UTR that holds 6 AUGs leading to several upstream open reading frames (uORFs). The initiation of translation from the 7th AUG forms a putative 55kDa precursor, which is processed in mitochondria to form a 52 kDa mature protein. The first three AUGs form fully overlapping uORF1, uORF2, and uORF3 that are in-frame with the 7th AUG and next two form fully overlapping uORF4 and uORF5 that are out-of-frame with the 7th AUG. Although not recognized by the scanning ribosomes under normal conditions, the 6th in-frame AUG forms a putative 57kDa extension of the main open reading frame. The purpose of this study was to identify the elements in the 5’UTR that direct CYP27 mRNA translation exclusively from the 7th AUG. Expression of 5' deletion mutants in COS cells reveal that the intact 5’UTR not only directs the initiation of translation from the 7th AUG but also acts as a negative regulator. A 2-kb deletion mutant that lacks uORF1 initiates translation equally from the 6th and the 7th AUGs forming both 57 kDa and 55 kDa precursor proteins with a two-fold increase in rate of translation. However, induction in translation does not affect the levels of mature 52 kDa form in mitochondria, but causes accumulation of the precursor form in cytosol not seen in COS cells transfected with wild-type cDNA. Mutation of the stop codon that terminates uORF1 completely shifts the initiation of translation from the 7th to the 1st AUG forming a 67-kDa precursor that is processed into a 52 kDa mature protein in mitochondria. Confirmation of the bicistronic nature of CYP27 mRNA by epitope mapping of uORF1 suggests that translation of CYP27 mRNA from the 7th AUG is directed and regulated by uORF1 expression.