The expression and localization of the proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G(4) AChE), were studied at vertebrate neuromuscular junctions. Both muscle and motor neuron contributed to this synaptic expression pattern. During the development of rat muscles, the expression of PRiMA and AChE(T) and the enzymatic activity increased dramatically; however, the proportion of G(4) AChE decreased. G(4) AChE in muscle was recognized specifically by a PRiMA antibody, indicating the association of this enzyme with PRiMA. Using western blot and ELISA, both PRiMA protein and PRiMA-linked G(4) AChE were found to be present in large amounts in fast-twitch muscle (e.g. tibialis), but in relatively low abundance in slow-twitch muscle (e.g. soleus). These results indicate that the expression level of PRiMA-linked G(4) AChE depends on muscle fiber type. In parallel, the expression of PRiMA, AChE(T) and G(4) AChE also increased in the spinal cord during development. Such expression in motor neurons contributed to the synaptic localization of G(4) AChE. After denervation, the expression of PRiMA, AChE(T) and G(4) AChE decreased markedly in the spinal cord, and in fast- and slow-twitch muscles.