Four experiments with C57BL/6 mice examined the effects of protein synthesis inhibition on extinction of spatial preferences in the Morris water maze. The protein synthesis inhibitor anisomycin was injected systemically immediately after spatial reversal sessions, which consisted of eight, two, or one reversal trials. Anisomycin had no effect on extinction of a spatial preference with eight reversal trials per session. When only two reversal trials were administered per session, anisomycin slowed extinction initially, although full extinction occurred with further training even in the presence of anisomycin. In both cases, anisomycin blocked acquisition of the reversal preference. When anisomycin injections followed a single simple extinction trial, performance was not disrupted. These findings suggest that acquisition and extinction may involve different molecular processes, and they do not support the idea that brief extinction trials induce a protein synthesis-dependent reconsolidation phase of spatial memory.