We studied phagolysosomal pH in alveolar macrophages (AM) using fluorescein-labeled yeast (FYP) and silica particles (FSP) as probes. Fluorescence intensities from the ingested test particles were measured on populations of AM using fluorescence spectrometry and on individual phagolysosomes using fluorescence microscopy. Measurements were performed on rabbit AM, which had been incubated with FYP or FSP (in vitro procedure). We also instilled FYP or FSP via the trachea into rabbit lungs and after 1 day, 1 week, 1 month, and 3 months lavaged the lungs and measured the pH in AM (in vivo procedure). Phagolysosomal pH was independent of the number and size of the fluorescent particles. Measurements of populations of AM with fluorescence spectrometry and of individual phagolysosomes with fluorescence microscopy gave similar average pH. For the FYP, pH decreased during the first day after lavage both in the in vitro and the in vivo procedures. For the FSP, pH was unchanged during the same period. After 1 day pH was similar for both particles. Electron microscopy showed a larger number of lysosomes in contact with phagosomes and a higher percentage of vacuolated phagosomes for FYP than for FSP. In the in vivo procedure, pH was unchanged at least up to 1 month, and this pH was lower than that in the in vitro procedure. The difference was probably due to conditions at the time of phagocytosis. Particles retained in the lung parenchyma were within AM, and their location within the AM appeared unchanged from 1 week up to 3 months.