<i>Objective:</i> During postnatal development, retinotectal projections undergo a process of misplaced axon elimination, leading to a topographical matching between the retinal surface and the superior colliculus. Matrix metalloproteinases (MMPs) have been implicated in the development and plasticity of the nervous system. We studied the expression and role of MMPs during normal development of retinotectal projections and after monocular enucleation-induced plasticity. <i>Material and Methods:</i> Lister hooded rats at different postnatal ages received subpial ethylene vinyl acetate 40W implants to deliver an MMP inhibitor or vehicle to the superior colliculus. Animals received intraocular injections of horseradish peroxidase for anterograde tracing of ipsilateral projections. For immunoblotting and zymography, colliculi were removed without fixation. <i>Results:</i> We observed the highest MMP activity in the first postnatal week, with decreasing activity thereafter. Monocular enucleation at postnatal day 10 yielded a rapid increase in MMP activity, 24 h following denervation of the contralateral colliculus. Importantly, inhibition of MMP activity in vivo induced a marked delay of axonal clustering along the medial aspect of colliculus. <i>Conclusions:</i> Our data indicate that MMPs are crucial in retinotectal development concurring to the fine tuning of topographical order and synaptic specificity of these connections.