In vivo the glomerular basement membrane is a specialised structure consisting of heparan sulphate proteoglycans (HSPG) and particular isoforms of collagen type IV and laminin. Currently there is controversy as to whether podocytes or glomerular endothelial cells (GEnC), or a combination of both, produce this matrix. We studied basement membrane production using conditionally immortalised human GEnC and podocyte cell lines. Both whole cell lysates and isolated basement membrane from conditionally immortalised cells in either the proliferating or differentiated state were analysed. Cells were removed from flasks or coverslips after incubation for 1 week, using 1% Triton containing 20mM ammonium hydroxide. This left behind basement membrane which was examined using immunofluorescence (IF) for pan collagen type IV, collagen IV α1, α2, α3 and α5 chains, heparan sulphate and binding of wheat germ agglutinin (lectin). The reactivity of the antibodies against glomerular basement membrane was confirmed by IF on tissue sections. The ability of cells to grow on matrix produced by cells was examined by light microscopy and IF for VE-cadherin. Cell and matrix lysates were further analysed by Western blotting for specific α chains of collagen type IV. IF (n=2) showed pan collagen type IV, collagen IV α1 and glycoproteins, including HSPG were produced by both cell types. Collagen IV α3 and α5 chains were detectable in proliferating and differentiated cells by Western blotting (n=3). Collagen IV α5 chains were only detectable in podocyte lysates, whereas α3 chains were present in both GEnC and podocytes lysates. Seeding GEnC onto podocyte produced matrix resulted in the cells growing normally, and vice versa. In summary, in vitro both cell types produce components of the specialised glomerular basement membrane which cells will grow on. Future work will focus on further analysis of the specific isoforms of collagen type IV produced by these cells, and also the rate at which these cells attach to matrix.