Intracellular distributions of the putative cytosolic pH indicator dyes BCECF [2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein], C.SNARF [5(and 6)-carboxy-seminaphthorhodafluor-1], and C.SNARF-calcein have been examined in Neurospora crassa and in murine fibroblasts (NIH-3T3 cells) under conditions in which both kinds of cells produce visible microscopic vacuoles. All three dyes were administered in electroneutral forms, with the hydroxyl and carboxyl groups esterified (designated as -AM esters). As judged qualitatively from fluorescence levels, hydrolytic derivatives of the two heavily esterified dyes (BCECF-AM and C.SNARF-calcein-AM) accumulated in the vacuoles after exposures of approximately 15 min or more, while the simpler dye (C.SNARF-AM) and its derivatives were almost excluded from visible vacuoles. Fluorescence from this dye, alone among the three, also washed out of Neurospora rapidly upon removal of extracellular dye. There was no evidence for stable accumulation of any of the dyes in cytosol per se. For BCECF(-AM), comparison of the distribution of fluorescence with the size distribution of vacuoles in Neurospora strongly suggests that the dyes are also accumulated by endomembranal vesicles (EMVs) which lie below the limit of resolution in the light microscope, and the same inference can be drawn for the fibroblasts. Uptake of -AM dyes by EMVs, including frank vacuoles, probably results from the action of intravesicular esterases, following diffusional entry of lipophilic neutral molecules or partially de-esterified anions. Calculations of actual cytosolic pH values, or even changes of pH, based on intracellular fluorescence of these dyes, clearly depend upon quantitative knowledge of the subcellular dye distribution. Therefore, until the problem is reliably solved of how to visualize submicroscopic vesicles in living cells, the safest approach to the use of BCECF, C-SNARF and their congeners for cytosolic pH measurement would be to devise methods for coaxing uptake of the ionic forms of these dyes and to abandon use of the esterified forms.