We report the use of the autofluorescent compound monodansylcadaverine (MDC) for in vivo labeling of autophagic vacuoles. When applied to various cell types (PaTu 8902, MDCK I, PC12, AR4-2J, WI-38) in culture, spherical structures were observed by fluorescence microscopy, predominantly located in the perinuclear region. Only PC12 and WI-38 cells had some of these labeled structures in their filopodiae. Dose-response experiments with PaTu 8902 showed that the optimal concentration for in vivo labeling was 0.05 to 0.1 mM, while cells detached and disintegrated, when MDC concentration exceeded 0.1 mM. After incubation with MDC and subcellular fractionations of PaTu 8902 cells on sucrose density gradients, a narrow fluorescence peak at 20 to 26% sucrose concentration equal to densities of about 1.081 to 1.108 g/cm3 was observed. Ultrastructural analysis of these fractions revealed autophagic vacuoles in different stages of their development. To investigate whether endosomal compartments were also labeled by MDC, we coincubated PaTu 8902 cells with MDC and the fluid-phase markers, RITC-dextran and ferritin, respectively. Fluorescence measurements after subcellular fractionations as well as fine structural analysis indicated that MDC-labeled autophagic vacuoles did not contain fluid-phase markers and were spatially separated from endosomal compartments. We further could demonstrate, after subcellular fractionation procedures, that MDC-labeled organelles contained the lysosomal enzymes acid phosphatase and the mature form of cathepsin D. Membrane markers of rough endoplasmic reticulum (TRAM and sec61 beta), and for smooth endoplasmic reticulum (cytochrome P450) were not detected in the same fractions. These results indicate that MDC accumulates as a selective fluorescent marker for autophagic vacuoles under in vivo conditions and is not present in the early and late endosome.