Some methodological aspects and characteristics on analysis of bone and liver alkaline phosphatase (EC 3.1.3.1, ALP) isoforms by high-performance liquid chromatography (HPLC) methods are presented. Factors affecting separation (analytical column type, column temperature, mobile phase buffer, mobile phase salt, gradient elution, flow rate) and reaction conditions (substrate type, substrate concentration, fluorescent substrates, substrate buffer type, substrate buffer concentration, substrate pH, substrate activators, substrate detergent, substrate flow rate, reaction temperature, postcolumn reaction detection) are discussed. Six peaks with ALP activity were separated and quantified by our new HPLC method in normal adult nonplacental serum: one intestinal/bone, two bone, and three liver ALP isoforms. Differences between the six ALP isoforms with respect to diethanolamine buffer concentration and substrate pH were observed. Although our HPLC method offers an improved possibility to clarify the reason for an increased total ALP in the routine clinical chemistry laboratory further research is needed to clarify the cellular origins of the different bone and liver ALP isoforms.