The biosynthetic pathway of pyridoxol (vitamin B(6)) in Rhizobium was clarified by studies on the incorporation of (13)C- or (15)N-labeled precursors into pyridoxol or its biosynthetic intermediates. Pyridoxol was formed by ring closure of two compounds, 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. The former was formed from D-glyceraldehyde and pyruvate through decarboxylation of pyruvate, and the latter from glycine and glycolaldehyde.