Tropomyosins (Tm) are a large family of isoforms obtained from multiple genes and by extensive alternative splicing. They bind in the alpha-helical groove of the actin filament and are therefore core components of this extensive cytoskeletal system. In non-muscle cells the Tm isoforms have been implicated in a diversity of processes including cytokinesis, vesicle transport, motility, morphogenesis and cell transformation. Using immunohistochemical localization in cultured primary cortical neurons with an antibody that potentially identifies all non-muscle TM5 gene isoforms compared with one that specifically identifies a subset of isoforms, the possibility was raised that there were considerably more isoforms derived from this gene than the four previously described. Using polymerase chain reaction (PCR) analysis we have now shown that the rat brain generates at least 10 mRNA isoforms using multiple combinations of terminal exons and two internal exons. There is extensive developmental regulation of these isoforms in the brain and there appears to be a switch in the preferential use of the two internal exons 6a to 6b from the embryonic to the adult isoforms. Specific isoforms using alternate carboxyl-terminal exons are differentially localized within the adult rat cerebellum. It is suggested that the tightly regulated spatial and temporal expression of Tm isoforms plays an important role in the development and maintenance of specific neuronal compartments. This may be achieved by isoforms providing unique structural properties to actin-based filaments within functionally distinct neuronal domains.