Changes in pH are measured in pinosomes and phagosomes of single specimens of the giant, free-living ameba, Chaos carolinensis. Measurements of pH are made microfluorometrically, as previously described (Heiple and Taylor. 1980. J. Cell Biol. 86:885-890.) by quantitation of fluorescence intensity ratios (Ex489nm,/Ex452nm, Ems2o-s6onm from ingested fluorescein thiocarbamyl (FTC)-ovalbumin. After I h of pinocytosis (induced in acid solution), FTCovalbumin is found in predominantly small (--<5 #m in diameter), acidic (pH -< 5.0-6.2) vesicles of various shape and density. As the length of ingestion time increases (up to 24 h), the probe is also found in vesicles of increasing size (up to 100 #m in diameter), increasing pH (up to pH -8.0), and decreasing density. Co-localization of fluorescein and rhodamine fluorescence, after a pulse-chase with fluorescein-and rhodamine-labeled ovalbumin, suggests vesicle growth, in part, by fusion. The pH in a single phagosome is followed after ingestion of ciliates in neutral solutions of FTC-ovalbumin. A dramatic acidification (A pH > -2.0) begins within 5 min of phagosome formation and appears to be complete in -20 min. Phagosomal pH then slowly recovers to more neutral values over the next 2 h. pH changes observed in more mature populations of pinosomes within a single cell may reflect those occurring within a single phagosome. Phagosomal and pinosomal pH changes may be required for lysosomal fusion and may be involved in regulation of lysosomal enzyme activity.Little is known about the regulation of intracellular vesicle movements after internalization of macromoleculcs or particles by endocytosis (30). Quantitative information about the normal sequence of ionic changes within endosomes is needed to understand the regulation of endosomal events such as endosome-lysosome fusion and the degradation or protection of internalized substances. Until recently, no satisfactory quantitative technique existed for the continuous measurement of pH within specific subcellular compartments of a single, moving eucaryotic cell. The novel microfluorometric technique we designed and applied to cytoplasmic pH measurements in single motile amebae (11) is here extended to a study of pH changes in pinosomes and phagosomes of these amebac.The free-living, fresh-water amebae, including Chaos carolinensis, are ideal cells for the study of a variety of fundamental processes. A great deal of quantitative information is available on both the physiology and morphology of free-living amebac (19) and extensive analysis of contractility and of endocytic mechanisms in these cells has been carried out over the last fifty years (7,30). The exceptionally large size of C. carolinensis