Publisher Summary Two β -galactosylpyridoxines and a β -galactobiosylpyridoxine are formed in high yield in a culture filtrate of Sporobolomyces singularis ( S. singularis ), when grown in a medium containing lactose and pyridoxine. All of the compounds are isolated as needle crystals and identified as 5'- O -( β -D-galactopyranosyl)pyridoxine, 4'- O -( β -D-galactopyranosyl)pyridoxine, and 4'- O -[ β -D-galactopyranosyl-(1→4)- O - β -D-galactopyranosyl] pyridoxine, respectively. Also, the yeast, when grown on a culture medium containing cellobiose and pyridoxine, produces a remarkable amount of 5'- O -( β -D-glucopyranosyl)pyridoxine and 4'- O -( β -D-glucopyranosyl)pyridoxine in a culture broth. S. singularis, when grown on a culture medium containing cellobiose and pyridoxine, forms 5'- O -( β -D-glucopyranosyl) pyridoxine and 4'- O -( β -D-glucopyranosyl)pyridoxine in the culture broth. These compounds are identified by comparison with the characteristics of 5'- O -( β -D-glucopyranosyl)pyridoxine and 4'- O -( β -D-glucopyranosyl) pyridoxine synthesized from cellobiose and pyridoxine by wheat bran β -glucosidase. The maximum yield of 4'- O -( β -D-glucopyranosyl)pyridoxine from lactose and pyridoxine by intact cells is observed at pH 4.0, while the yield of 5'- O -( β -D-glucopyranosyl) pyridoxine is observed at pH 5.5. Lactose, o -nitrophenyl β -D-galactopyranoside, cellobiose, salicin, phenyl β -D-glucopyranoside, and p -nitrophenyl β -D-glucopyranoside effect the formation of glycosylpyridoxine, but galactose, glucose, glucose 1-phosphate, melibiose, raffinose, maltose, and sucrose are quite ineffective.