Vascular endothelial growth factor recpetor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and -D (VEGF-D), are the major molecules involved in developmental and pathological lymphangiogenesis. Here we describe for the first time the development of a specific indirect enzyme-linked immunosorbent assay (ELISA) for the quantification of VEGFR-3 in different human cell and tissue lysates. A combination of the goat polyclonal anti-VEGFR-3 antibody and the mouse monoclonal anti-human VEGFR-3 antibody was used. The assay was highly sensitive and reproducible with a detection range of 0.2-25 ng/ml. The assay was specific for VEGFR-3, with no cross-reactivity to VEGFR-1 or VEGFR-2. Complex formation with VEGF-C and VEGF-D had no effect on the sensitivity of the assay. The VEGFR-3 concentration in the lysates of cultured human dermal microvascular endothelial cells was 14-fold higher than in the lysates from human umbilical vein endothelial cells. In human kidney, breast, colon, gastric and lung cancer tissues the protein levels of VEGFR-3 were in the range of 0.6-16.7 ng/mg protein. Importantly, the level of VEGFR-3 protein detected in the ELISA correlated significantly with the number of VEGFR-3 positive vessels observed in histochemical sections, suggesting that the ELISA assay may be a reliable surrogate of measuring VEGFR-3-positive vessel density. The protein levels of VEGFR-3 in 27 renal cell carcinoma samples had a significant correlation with the levels of VEGF-C (p<0.001), or biological active, free VEGF-A (p<0.0001), but not with VEGFR-1 or total VEGF-A. This assay provides a useful tool for the investigations of the expression levels of VEGFR-3 in physiological and pathological processes, particular in cancer and in lymphangiogenesis-related disease. (C) 2004 Wiley-Liss, Inc.