The enzyme serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in the de novo synthesis of sphingolipids. Previously the mammalian SPT was described as a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Recently we identified a novel third SPT subunit (SPTLC3). SPTLC3 shows about 68% identity to SPTLC2 and also includes a pyridoxal phosphate consensus motif. Here we report that the overexpression of SPTLC3 in HEK293 cells leads to the formation of two new sphingoid base metabolites, namely C(16)-sphinganine and C(16)-sphingosine. SPTLC3-expressing cells have higher in vitro SPT activities with lauryl- and myristoyl-CoA than SPTLC2-expressing cells, and SPTLC3 mRNA expression levels correlate closely with the C(16)-sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C(16) backbone and are found in the high density and low density but not the very low density lipoprotein fraction. In conclusion, we show that the SPTLC3 subunit generates C(16)-sphingoid bases and that sphingolipids with a C(16) backbone constitute a significant proportion of human plasma sphingolipids.