Synthesis of the ceramide portion of sphingolipids in animals has been hypothesized to be tightly regulated thereby controlling the rate of de novo sphingolipid formation. Regulation is predicted to occur at the first and committed biosynthetic step catalyzed by serine palmitoyltransferase (SPT, EC 2.3.1.50). This hypothesis remains unproven because SPT has been refractory to purification and subsequent characterization. To begin to test this hypothesis we have used a genetic strategy to isolate LCB2 homologs from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe and a cDNA homolog from humans and mice. Identity is supported by overall amino acid sequence similarity between the predicted proteins and the known Saccharomyces cerevisiae Lcb2 protein. In addition, a motif of 56 residues from the human protein functionally substituted for the corresponding region of the S. cerevisiae Lcb2 protein. The 56 residue motif was found to be unique to Lcb2 proteins. Likewise, the base sequence encoding it is unique to the human genome. Finally, a peptide sequence in the motif is known to be part of the catalytic domain of all members of the aminolevulinate synthase superfamily of proteins of which Lcb2 is a member. These data argue that this motif is part of the catalytic domain of SPT and is a signature of Lcb2 proteins. The mammalian LCB2 cDNAs provide valuable reagents for studying the Lcb2 subunit of SPT and for studying how ceramide synthesis is regulated.