A high performance liquid chromatographic (HPLC) system consisting of a binary mobile phase, a µBondapak C₁₈ column, and a fluorescence detector was used to separate the B-6 vitamers pyridoxal (PL), pyridoxine CPN), and pyridoxamine (PM) as well as the internal standard deoxypyridoxine. An extraction procedure, compatible with the HPLC system, was developed to isolate the B-6 vitamers from rat livers, kidneys, and brains. PL values were derived from the sum of tissue PL and pyridoxal phosphate (PLP), the latter having been dephosphorylated to PL. Similarly, PN values were the sum of tissue PN and pyridoxine phosphate (PNP) and PM data the sum of PM and pyridoxamine phosphate (PMP). Separation and quantitation of tissue vitamers were successful and reflected significant relationships between rat vitamin B-6 intakes and tissue concentrations of the B-6 vitamers PL and PM as well as total B-6 vitamer concentration. PM was found to be the major vitamer in all tissues. The HPLC data were compared to values obtained by the AOAC microbiological (MB) assay performed on the same samples. Significant correlations (r) ranging from 0.31 - 0.73 for PL, 0.30 - 0.47 for PM, and 0.48 - 0.55 for total B-6 vitamer concentration were observed between values obtained via the methods. Both HPLC and MB vitamer data approximated reported values. The HPLC method offered the advantages of relative speed and simplicity. / M.S.