Despite intensive research over the past decade, the exact lineage relationship of Kaposi's sarcoma (KS) tumor cells has not yet been settled. In the present study, we investigated the expression of two markers for lymphatic endothelial cells (EC), ie, vascular endothelial growth factor receptor-3 (VEGFR-3) and podoplanin, in AIDS and classic KS. Both markers were strongly expressed by cells lining irregular vascular spaces in early KS lesions and by tumor cells in advanced KS. Double-staining experiments by confocal laser microscopy established that VEGFR-3-positive and podoplanin-positive cell populations were identical and uniformly expressed CD31. By contrast, these cells were negative for CD45, CD68, and PAL-E, excluding their hemopoietic and blood vessel endothelial cell nature. Podoplanin expression in primary KS tumor lysates was confirmed by Western blot analysis. Both splice variants of VEGFR-3 were found in KS-tumor-derived RNA by RT-PCR. In contrast to KS tumor cells in situ, no expression of VEGFR-3 and podoplanin was detected in any of four KS-derived spindle cell cultures and in one KS-derived autonomously growing cell line (KS Y-1). Our findings that KS tumor cells express two lymphatic EC markers in situ strongly suggest that they are related to or even derived from the lymphatic EC lineage. Lack of these antigens on cultured cells derived from KS lesions indicates that they might not represent tumor cells that grow in tissue culture, but rather other cell types present in KS lesions.