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, Em520-560nm from ingested fluorescein thiocarbamyl (FTC)-ovalbumin. After 1 h of pinocytosis (induced in acid solution), FTC-ovalbumin is found in predominantly small ( less than or equal to 5 micrometers in diameter), acidic (pH less than or equal to 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 micrometers in diameter), increasing pH (up to pH approximately 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 (delta pH greater than or equal to - 2.0) begins within 5 min of phagosome formation and appears to be complete in approximately 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.