A simple, accurate reverse-phase high performance liquid chromatography (RPLC) method was introduced for the analysis of B-6 vitamers in human milk. The assay consisted of a phosphate buffer (pH 2.9) delivered isocratically through 5 micron ODS column packing, followed by post-column bisulfite derivatization to enhance the detection of PLP (and to a minor extent PL). The vitamers were detected using a fluorescence spectromonitor. Sample run time was less than 30 min. The sensitivity of the method was such that PL, PN, and PLP were detectable to 30 pmol/ml milk and PM and PMP to 5 pmol/ml milk. Total vitamin B-6 content in milk analyzed by RPLC correlated well with the microbiological assay. B-6 vitamer distribution in human milk was similar to values obtained from two different ion-exchange HPLC systems. The RPLC procedure is simpler and faster than the HPLC systems and is suggested for future use in analysis of B-6 vitamer concentrations in human milk.