The fatty acid desaturase (Fads) cluster is composed of three genes encoding for the Δ5- and Δ6-desaturases and FADS3. The two former proteins are involved in the fatty acid biosynthesis; the latter one shares a high sequence identity but has still no attributed function. In a previous work performed in rat, we described three isoforms of FADS3 expressed in a tissue-dependent manner. In the present study, we demonstrated a specific subcellular targeting depending on the isoform. In cultured hepatocytes, which mainly expressed the 51 kDa protein, FADS3 was unexpectedly present in the cytosolic fraction, but was also secreted in the extracellular matrix on fibronectin-containing fibers. The secretion pathway was investigated and we determined the presence of exosome-like vesicles on the FADS3-stained fibers. In parallel, FADS3 was detected in blood of hepatic vessel, and particularly in serum. In conclusion, this study demonstrated a very specific intra- and extracellular location of FADS3 in comparison with the Δ5- and Δ6-desaturases, suggesting a unique function for this putative desaturase, even if no activity has been yet identified neither in the extracellular matrix of hepatocytes nor in serum. J. Cell. Biochem. 115: 199–207, 2014. © 2013 Wiley Periodicals, Inc.