A weak anion-exchange high-performance liquid chromatographic procedure with post-column reaction detection for simultaneous determination of alkaline phosphatase (EC 3.1.3.1, ALP) isoenzymes is described. We identified six peaks with ALP isoenzyme activity in normal serum. The peaks were, in order of elution, one intestinal/bone, two bone and three liver ALP isoenzymes. This new assay with automatic injection, on-line post-column reaction detection and powerful integration data system could be of significant value in the routine clinical biochemistry laboratory. The advantages include improved sensitivity and selectivity over previous methods for the determination of ALP isoenzymes.