We have cloned the cDNA for a GD3 synthase (α-2, 8-sialyltransferase, [EC 2. 4. 99. 8]) from a rat embryonic brain cDNA library. Mammalian cells transfected with the cloned cDNA expressed GD3 on the cell surface and showed GD3 synthase activity. The deduced protein (342 amino acid residues) was predicted to have a type II membrane topology containing the “sialyl motif” and was found to be 91% similar to its human homologue. Analysis of the acceptor specificity of GD3 synthase protein indicated that this enzyme catalyzed the biosynthesis of GT1a and GQ1b as well as GD3. Northern blot analyses showed that the GD3 synthase gene is preferentially transcribed in the brain and the spleen. The expression of GD3 synthase mRNA was developmentally regulated, with the highest level in the brain during embryonic days 15 to 18. In situ hybridization analyses demonstrated that the GD3 synthase is strongly expressed in the ventricular/subventricular zone of the embryonic rat brain and retina. In the adult rat, GD3 synthase mRNA was detected in the cerebral cortex, hippocampus, thalamus, and cerebellum. These studies show that the spatio- and stagerestricted expression of GD3 in the developing rat brain may be regulated in part by the level of GD3 synthase mRNA.