It is shown by incorporation experiments with 13C bond-labeled substrates, followed by analysis by means of 13C NMR spectroscopy, that two compounds, 1-deoxy-D-xylulose (12) and 4-hydroxy-L-threonine (), serve as precursors of pyridoxol (vitamin B6) (1) in Escherichia coli. Together, these two compounds account for the entire C8N skeleton of the vitamin. 1-Deoxy-D-xylulose supplies the intact C5 unit, C-2',2,3,4,4' of pyridoxol. 4-Hydroxy-L-threonine undergoes decarboxylation in supplying the intact C3N unit, N-1,C-6,5,5'. Both precursors are ultimately derived from glucose. The C5 unit of pyridoxol that is derived from 1-deoxy-D-xylulose originates by union of a triose phosphate (yielding C-3,4,4') with pyruvic acid (which decarboxylates to yield C-2',2). D-Erythroate (11) enters the C3 unit, C-6,5,5', and is therefore an intermediate on the route from glucose into 4-hydroxy-L-threonine.