Conventional fluorescence microscopy of fixed HEp-2 cells as well as video microscopy of living cells incubated with transferrin-Texas Red (Tf-TxR) for < 60 min revealed distinct punctuate endosomal structures. Quantitative ultrastructural analysis using horseradish peroxidase (HRP) and cationized gold as tracers showed that spherical multivesicular bodies (MVBs) were the predominant endocytic compartments in HEp-2 cells and that MVBs within 60 to 90 min matured into lysosomes still containing internal vesicles. The number of labeled MVBs increased continuously from 2.5 min to 30 min of tracer incubation. However, when the cells were pulsed for 5 min followed by 10 or 25 min chases, the number of labeled MVBs corresponded to that obtained after 5 min of continuous incubation. The diameter of labeled MVBs was largely constant with time, but the number of internal MVB vesicles increased. Thus, early or newly formed MVBs contained few internal vesicles, whereas late MVBs, that is to say, MVBs that have existed for some period of time, contained numerous internal vesicles, and finally a mixture of membranous material or myelin figures and vesicles. It is thus in principle possible to distinguish between early and late MVBs in HEp-2 cells on the basis of morphology. However, the difference in number of internal vesicles applies only to the entire MVB population; after only 2.5 to 5 min of incubation, MVBs with numerous internal vesicles could also be reached by internalized tracer. Concomitant with the gradual changes in morphology, the MVBs also showed a characteristic change in content of marker proteins as detected by immunogold labeling on ultracryosections. Hence, early MVBs with relatively few internal vesicles and typically reached by internalized tracers within 5 min contained transferrin receptors (TfRs). By contrast, MVBs with many internal vesicles and labeled after 60 min of incubation contained mannose-phosphate receptors (MPRs), and the MVBs with distinct membranous material or myelin figures in addition to the internal vesicles were enriched in the lysosome membrane protein lamp-1. Thus, there seems to be a gradual maturation of MVBs in HEp-2 cells.