Our objective was to correlate biochemical or functional properties that could be measured by fluorescence to individual nonparenchymal cell types by the simultaneous detection of forward angle light scatter and fluorescence. Cells were released from liver following selective digestion of hepatocytes by Pronase perfusion. To fractionate the cells according to size, isolates were subjected to unit gravity sedimentation on a 1 to 5% sucrose gradient. Flow cytometry was used to analyze the resulting fractions as well as the original unfractionated cells. This reaffirmed that forward angle light scatter is linearly related to the sedimentation velocity (size) of the cells and that it can be used to assess cell sizes in an unfractionated population. Endocytosis of fluorescently tagged particulates was studied by injecting either fluorescein isothiocyanate-tagged colloidal albumin or Coumarin-tagged latex beads (0.57 micron diameter) prior to Pronase perfusion. Beads were found only in the largest cells (Kupffer cells). Since endothelial cells could endocytose only colloidal albumin, they were readily differentiated from nonendocytosing, nonsinusoidal cells ("lymphocytes"). Staining cell protein with fluorescein isothiocyanate made possible the determination of relative protein content per cell ("lymphocyte": endothelial:Kupffer cell was 0.57:1.0:2.0). To determine relative esterase activities, mixed cell isolates were incubated in fluorescein diacetate ("lymphocyte":endothelial:Kupffer cells was 0.13:1.0:2.4). Uptake of rhodamine 123 was used to assess mitochondrial function ("lymphocyte":endothelial:Kupffer cells was 0.45:1.0:14.6). Mitochondrial volume per cell is known to be 1:2 for endothelial:Kupffer cells. Therefore, the large difference in rhodamine uptake is not related to volume.