Human neutrophils sequester yeast cells or zymosan particles in two classes of phagocytic vacuoles: sealed and unsealed. The pH of sealed vacuoles was measured by a newly devised fluorometric procedure that used fluoresceinated zymosan particles, derived from Saccharomyces cerevisiae, as a pH probe. Five minutes after initiation of phagocytosis, sealed vacuoles were alkaline, exhibiting a pH of 7.80 +/- 0.19 (mean +/- SEM). Their acidity subsequently increased, so that the pH fell to 7.38 +/- 0.25 after 15 min. 6.35 +/- 0.35 after 30 min, and 5.68 +/- 0.26 after 60 min. The implications of these findings for neutrophil microbicidal mechanisms are discussed.