The degradation of the extracellular matrix is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Matrix components of the basement membrane play critical roles in the development and maintenance of the neuromuscular junction (NMJ), yet almost nothing is known about the regulation of MMP and TIMP expression in either the presynaptic or postsynaptic compartments. Here, we demonstrate that TIMP-2 is expressed by both spinal motor neurons and skeletal muscle. To determine whether motor function is altered in the absence of TIMP-2, motor behavior was assessed using a battery of tests (e.g., RotaRod, balance beam, hindlimb extension, grip strength, loaded grid and gait analysis). TIMP-2−/− mice fall off the RotaRod significantly faster than wild-type littermates. In addition, hindlimb extension is reduced and gait is both splayed and lengthened in TIMP-2−/− mice. Motor dysfunction is more pronounced during early postnatal development. A preliminary analysis revealed NMJ alterations in TIMP-2−/− mice. Juvenile TIMP-2−/− mice have increased nerve branching and acetylcholine receptor expression. Adult TIMP-2−/− endplates are enlarged and more complex. This suggests a role for TIMP-2 in NMJ sculpting during development. In contrast to the increased NMJ nerve branching, cerebellar Purkinje cells have decreased neurite outgrowth. Thus, the TIMP-2−/− motor phenotype is likely due to both peripheral and central defects. The tissue specificity of the nerve branching phenotype suggests the involvement of different MMPs and/or extracellular matrix molecules underlying the TIMP-2−/− motor phenotype.