The kinetics of acidification of diferric human transferrin in BALB/c mouse 3T3 cells were determined by flow cytometry using a modification of the fluorescein-rhodamine fluorescence ratio technique. For cells labeled at 0 degrees C and warmed to 37 degrees C, the minimum pH observed was 6.1 +/- 0.2, occurring 5 min after warmup. This step was followed by a slower alkalinization to the pH of the external medium, occurring with a half-time of 5 min. Warmup to 24 degrees C or 17 degrees C resulted in slowing of the time of onset of acidification such that the minimum pH was 6.3 +/- 0.2, attained 15 and 25 min after warmup, respectively; the alkalinization step was completely blocked. The limited acidification observed for transferrin corresponds to the initial phase of acidification normally observed for other (nonrecycled) ligands. Since transferrin is not further acidified, the results confirm the existence of two phases of acidification during endocytosis. Measurements of transferrin dissociation at neutral pH after exposure to mildly acidic pH support the conclusion that the transferrin cycle may be completed without exposure of transferrin to a pH below 6. The mildly acidic pH of the endocytic compartments involved in recycling may play a role in regulating enzymatic processing of endocytosed material.