Axonal elongation, neurite outgrowth, and synaptogenesis are key events of mammalian brain development. The neuronal cytoskeleton plays a fundamental role in these changes, which are influenced by gonadal steroids in brain regions bearing the appropriate receptors. Microtubules, which constitute a major portion of the cytoskeleton, are composed of alpha- and beta-tubulin polypeptide heterodimers encoded by multigene families. Of the five beta-tubulin isotypes described, the genes encoding Class II and IV are preferentially expressed in brain. Expression of the mRNA encoding Class II beta-tubulin isotype is selectively increased during neuronal development and regeneration, whereas that of Class IV increases later in development and does not change during regeneration. The present study was undertaken to characterize the expression of these mRNAs during hypothalamic development and to determine if the well-known process of steroid-dependent sexual differentiation of this brain region is accompanied by related differences in beta-tubulin mRNA expression. In addition, we examined the developmental profile of mRNAs encoding two protein constituents of neurofllaments (NF), the other major component of the neuronal cytoskeleton. The levels of Class II beta-tubulin mRNA (RBTZ) were elevated in the medial basal hypothalamus (MBH) and preoptic area (POA) of both males and females during the late fetal-early postnatal period (i.e., Fetal Day 16-Postnatal Day 2) and decreased markedly thereafter. The initial decrease, detected between Postnatal Days 2 and 12, was more pronounced in females than in males, the difference being more clear in the POA than in the MBH. No such sexrelated differences were found in the cerebral cortex (Cc) or cerebellum (Cb). Castration of male rats on the day of birth reduced RBTZ mRNA levels in the POA and MBH toward female values when measured on Day 12. Analysis of the Class IV beta-tubulin mRNA (RBT(2)) developmental profile revealed that RBTZ mRNA is expressed throughout development (Fetal Day 20-Postnatal Day 25) in the POA and MBH, without sex-related differences. Although levels of expression increased during the fourth week of life, the increase was less conspicuous than in the Cb. Levels of the two mRNAs encoding the 68-kDa protein of neurofilaments (NF68) increased moderately between Fetal Day 16 and the day of birth in both the POA and MBH, remaining relatively constant thereafter, without detectable sex differences. Levels of NF145 mRNA, which encodes the 145-kDa protein of neurofilaments, were already maximally elevated by Fetal Day 16 (the earliest age studied) and showed no sex-related differences during development. Since RBTZ, but not RBT(2) or NF, mRNA expression is known to be induced during neuronal development, the results provide molecular evidence for the concept that an extended period of axonal elongation/neurite outgrowth underlies the steroid-dependent morphological differentiation of the rat hypothalamus.