Hyalocytes, the cells of the vitreous body, are assumed to be involved in physiological as well as patho-physiological processes within the eye. However, current knowledge about the cells is still limited. As different morphological types of hyalocytes are described in the literature, it seems reasonable to try to isolate individual populations prior to characterization of single cell types. To achieve this, the present study investigated the utility of fluorescence activated cell sorting (FACS) for hyalocyte separation. Subsequent to digestion of vitreous bodies using collagenase, the resulting cell suspension was analyzed and separated using FACS without any additional staining. Two-parameter dot plots of forward scatter (indicating size) against sideward scatter (indicating granularity) showed two distinct cell populations; staining with propidium iodide confirmed that both populations represent living cells. After sorting, cells of both populations were seeded on tissue culture plastic (tissue culture treated polystyrene). Only one population attached and proliferated, whereas the other population was non-adherent. Even when seeding the native cell mix, only one population of cells was observed after two passages, as indicated by FACS. Furthermore, ascorbic acid increased proliferation of these cells similarly to the proliferation of the separated cell population. These data point out that only one of the two populations adheres and proliferates on tissue culture plastic. To conclude, the established isolation technique allows for separation of clearly defined hyalocyte populations. Moreover, clear hints were obtained that only one of the two populations adheres and proliferates under the commonly applied culture conditions.