Escherichia coli KG980, a vitamin B6 auxotroph derived from E. coli K12, utilized the three unphosphorylated forms of vitamin B6, more or less effectively, for growth, but did not utilize the three phosphate forms at concentrations ranging from 10(-7) to 10(-5) M in the minimum medium of Davis and Mingioli. The bacterium, however, utilized the phosphate forms, although less effectively than the unphosphorylated forms, in the phosphate starving medium of Garen and Levinthal which is known to derepress alkaline phosphatase. Correspondingly, the phosphate forms of 3H-labeled vitamin B6 in the minimum medium were not taken up by the bacterial cells grown in the same medium, but were taken up when the phosphate starving medium was used for growth and uptake experiments; the unphosphorylated forms were taken up with either of the media used. After 30-min incubation of the cells grown in the phosphate starving medium with 3H-pyridoxine phosphate added to the same medium, the main extracellular 3H-vitamin B6 was found to be pyridoxine, evidently indicating an involvement of alkaline phosphatase action. It is concluded from these results that the phosphate forms of vitamin B6 can be taken up and utilized only after dephosphorylation but not taken up in their intact form. The initial rate of 3H-pyridoxal and 3H-pyridoxamine uptake in the minimum medium showed saturation kinetics. The Km and Vmax values were 1.2 X 10(-6) M and 62 pmoles/min/mg (dry cell weight) for pyridoxal; 11 X 10(-6) M and 65 pmoles/min/mg for pyridoxamine. 3H-Pyridoxine uptake apparently consisted of a high-affinity saturable component, whose Km value was tentatively estimated to be 2.2 X 10(-6) M, and an unsaturable component. The uptake rates of these three unphosphorylated vitamin B6 compounds compared at limiting concentrations for the growth of E. coli KG980 appear to be essentially in good agreement with the response pattern of this bacterium to the three compounds.