The lysosomal pH in Mucolipidosis type IV (ML-IV) and several other storage disease fibroblasts (Niemann Pick, type A; Niemann Pick, type C; Hunter (MPS II); and Farber) and in normal human skin fibroblasts was determined in situ. Cells were pulse labeled with a fluorescein-conjugated dextran to label the lysosomes. Quantitative fluorescence microscopy was then carried out on living cells to measure the ratio of fluorescence at two different excitation wavelengths. An image processing routine was used to quantify fluorescence from individual lysosomes. Ratiometric data were converted to an absolute value of pH using an appropriate standard curve. Lysosomal pH varied between 4.3 and 4.5 for all the cell types examined except ML-IV cells which was almost one pH unit higher (pH approximately 5.2). Qualitatively similar results were obtained using acridine orange, another fluorophore whose fluorescence emission is pH dependent, ruling out the possibility that the stored molecules in ML-IV cells might induce an artifact in the fluorescein-based pH measurements. We conclude that elevated lysosomal pH is unique to ML-IV cells. This property may be an important factor, if not the cause, for the accumulation of the broad spectrum of substances, including sphingolipids, phospholipids, and acid mucopolysaccharides, even though the lysosomal hydrolases participating in the catabolism of these molecules appear to be normal.