Doxycycline, a broad-spectrum antibiotic, has certain antiangiogenic properties and can inhibit matrix metalloproteinases (MMPs/gelatinases). The authors investigated the effects of doxycycline on choroidal neovascularization (CNV) and regulation of MMP-2 and -9 and pigment epithelium-derived factor (PEDF). Doxycycline was orally administered to rats at 500, 50, 5, and 0.5 mg/kg/d; nontreated animals were used as controls. Experimental CNV was induced with laser 7 days after doxycycline treatment started. At 7 days after induction, animals were euthanatized, and eyes were collected. RPE/choroid flatmounts were labeled with isolectin IB4 to determine CNV lesion volumes using confocal microscopy and high-performance 3D imaging software. MMP-2, MMP-9, and PEDF protein levels were determined by ELISA. MMP catalytic activity was determined in solution using fluorogenic gelatin and peptide substrates, by gelatin zymography in SDS-PAGE, and by in situ fluorogenic substrate zymography in RPE/choroid sections. CNV complex lesion volumes decreased with doxycycline in a dose-response relationship. A dosage of 500 mg/kg/d caused a 70% inhibition of CNV complex volume compared with control animals. Doxycycline elevated PEDF levels in plasma and did not affect the active and pro-enzymes MMP-2 and MMP-9 levels. However, the in vitro enzymatic activities of purified MMP-2 and MMP-9 declined significantly with doxycycline. MMP-2, MMP-9, and gelatinolytic activities in situ increased early in CNV lesion development. Doxycycline treatments and exogenous additions inhibited gelatinolytic activities in CNV lesions. Doxycycline effectively hampered the progression of experimental CNV. The results suggest that orally administrated doxycycline can reach the choroid to attenuate proteolytic enzymes that remodel Bruch's membrane and promote the antiangiogenic PEDF to inhibit neovascularization.