Using a combination of L-phenylalanine inhibition and heat inactivation, the serum alkaline phosphatase (AP) in 2 to 13 year old children without evidence of hepatobiliary, osseous, or intestinal disease was separated in three fractions; i.e. L-phenylalanine sensitive AP (LPSAP), heat-stable non-L-phenylalanine sensitive AP (heat-stable non-LPSAP) and heat sensitive non-L-phenylalanine sensitive AP (heat-sensitive non-LPSAP). The activities of total AP and the different fractions were measured using optimized test conditions. LPSAP, (mainly intestinal AP), accounts for approximately 12% of the total serum AP activity, heat-stable non-LPSAP (mainly hepatobiliary AP) for approximately 9%, and heat-sensitive non-LPSAP (mainly bone AP) for approximately 77%. To give a better differentiation between bone and liver AP, the percentage ratios of heat-stable non-LPSAP/non-LPSAP (Q value), and heat-stable non-LPSAP/total AP, were determined. Both quotients showed a significant negative correlation with total AP, which has to be taken into account in the interpretation of the results of isoenzyme determinations of serum AP activity. The above semiquantitative separation of AP isoenzymes can be readily done in a routine clinical laboratory.