Regulated proteolysis by the ubiquitin pathway has been implicated in control of the cell cycle, transcriptional activation, cell fate and growth, and synaptogenesis. The ubiquitin-proteasome system is involved in synaptic plasticity and is proposed to be part of a molecular switch that converts short-term synaptic potentiation to long-term changes in synaptic strength. In Aplysia, a component of the ubiquitin system termed ubiquitin C-terminal hydrolase (Ap-Uch) has been shown to be essential for long-term facilitation. To examine whether Uch plays a role in learning, memory, and synaptic plasticity in mammals, we have analyzed mice homozygous for a targeted mutation in ubiquitin C-terminal hydrolase L3 (Uchl3), an orthologue of Ap-Uch. Mice homozygous for the mutation in Uchl3 are viable, with no obvious developmental, histological, or fertility abnormalities. We demonstrate that Uchl3-/- mice have a significant learning deficit relative to wild type littermates in the spatial version of the Morris water maze and the 8-arm radial maze. Further, the impaired performance in the 8-arm radial maze of Uchl3-/- mice is due to significantly increased working memory errors. Examination of hippocampal long-term potentiation (LTP), a form of synaptic plasticity thought to underlie memory storage, revealed no significant differences in LTP in hippocampal slices from Uchl3-/- mice. Our results suggest a novel role for ubiquitin C-terminal hydrolase enzymes in mammals in spatial learning and working memory.