PURPOSE. The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch's membrane are altered in Efemp1(ki/ki) mice carrying this mutation or in Efemp1(-/-) mice. METHODS. Proteoglycans in mouse Bruch's membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch's membrane/choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (R-s) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. RESULTS. HSPGs and C/DSPGs were markedly increased in Efemp1(ki/ki) Bruch's membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1(ki/ki) Bruch's membrane was impaired. In contrast, the proteoglycan amount in Efemp1(-/-) Bruch's membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1(+/+) mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch's membrane. Diffusion across Efemp1(-/-) Bruch's membrane was enhanced. CONCLUSIONS. Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch's membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch's membrane.