Our objectives were to evaluate kinase insert domain protein receptor (KDR)-β-galactosidase (LacZ) expression as a marker for vascular development during gonadal morphogenesis and to determine whether any novel non-angiogenic KDR-LacZ expression was present in mouse testes or ovaries. Gonads were collected from mice expressing LacZ driven by the Kdr promoter (KDR-LacZ) from embryonic day 11 (E11) through postnatal day 60 (P60). At E11.5, mesonephric cells expressing KDR-LacZ seemed to migrate into the developing testis and surrounded developing seminiferous cords. Cells expressing KDR-LacZ appeared in the ovary with no apparent migration from the adjacent mesonephros, suggesting a different origin of endothelial cells. Testis organ cultures from E11 mice were treated with 8 μM VEGFR-TKI, a vascular endothelial growth factor A signal transduction inhibitor; subsequently, the amount of KDR-LacZ staining was reduced by 66%-99% (P<0.002), and the ability of KDR-expressing cells to form a densely organized vascular network was inhibited. Novel non-angiogenic KDR-LacZ staining was detected in the testis on specific subsets of germ cells at E16, E17, P4, P20, P30, and P60. In ovaries, staining was present on oocytes within oocyte cysts at E17 and within late secondary follicles of postnatal mice. Thus, KDR is an excellent marker for analyzing vascular development in the gonads. Inhibition of VEGFA signal transduction prevents the development of testis-specific vasculature. Furthermore, non-vascular KDR-LacZ staining suggests that KDR directly affects both spermatogenesis and somatic-oocyte interactions during gametogenesis.