Therapeutic regulation of tissue vascularization has appeared as an attractive approach to treat a number of human diseases. In vivo neovascularization assays that reflect physiological and pathological formation of neovessels are important in this effort. In this report we present an assay where the effects of activators and inhibitors of angiogenesis can be quantitatively and qualitatively measured. A provisional matrix composed of collagen I and fibrin was formed in a plastic cylinder and implanted onto the chick chorioallantoic membrane. A nylon mesh separated the implanted matrix from the underlying tissue to distinguish new from pre-existing vessels. Vascularization of the matrix in response to fibroblast growth factor-2 or platelet-derived growth factor-BB was scored in a double-blinded manner, or vessel density was measured using a semi-automated image analysis procedure. Thalidomide, fumagillin, U0126 and TGF beta inhibited neovessel growth while hydrocortisone exerted a negative and wortmannin a toxic effect on the pre-existing vasculature. This quantitative, inexpensive and rapid in vivo angiogenesis assay might be a valuable tool in screening and characterizing factors that influence wound or tumor induced vascularization and in assessing their effects on the normal vasculature.