Neuronal differentiation involves extensive rearrangement of the cytoskeleton, including the actin-based microfilament system, and establishment of molecular compartments within the neuron. The intracellular distribution of tropomyosin (Tm) mRNA in vivo and in vitro has been examined and correlated with protein targetting. The mRNAs encoding two Tm isoforms were found to be differentially localized in developing neurons. Tm-5 mRNA is localized to the axonal pole of differentiating embryonic rat neurons, in contrast to TmBr-2 mRNA distribution throughout the cell body. Tm-5 mRNA is transported into the axon of differentiating primary cultured neurons. This mRNA localization is developmentally regulated and correlates with the targeting of Tm-5 protein to growing axons. Tm-5 colocalizes with a subset of neuronal microfilaments associated with the initiation and maintenance of outgrowth. The segregation of Tm-5 is the earliest known marker of neuronal polarity and may play a role in the establishment of polarity.