Isoenzymes of alpha-glucosidases (EC 3.2.1.20) from various human organs and body fluids from fetuses and adults were separated by DEAE-cellulose column chromatography and gel filtration using Sephadex G-100. A minicolumn (0.35 X 2.5 cm) was used for the DEAE-cellulose column chromatography of extracts from tissues as well as cultivated cells of skin fibroblasts and amniotic fluid. The enzyme activity in the eluates was measured by the use of a methylumbelliferyl derivative as substrate and a very sensitive Microscope fluorimeter. In most tissue samples alpha-glucosidase was eluted mainly as a single peak when monitored at acid pH and as two peaks when the activity was measured at neutral pH in both columns. Another small peak representing alpha-glucosidase was found in fresh extracts of cultured cells on DEAE-cellulose columns. Neutral alpha-glucosidase especially in fibroblasts was extremely sensitive to storage at -20 degrees C. DEAE-cellulose column chromatography of plasma and amniotic fluid showed similar elution patterns of alpha-glucosidase. Differences were noticed in the elution pattern of urine from infants and adults. The tissue distribution and the different characteristics of the enzyme in samples of various origins and ages were discussed.