Five β‐tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene cβ₄ and has been assigned to an isotypic family designated as Class III (βIII). In the nervous system of higher vertebrates, βIII is synthesized exclusively by neurons. A βIII‐specific monoclonal antibody was used to determine when during chick embryogenesis cβ₄ is expressed, the cellular localization of βIII, and the number of charge variants (isoforms) into which βIII can be resolved by isoelectric focusing. On Western blots, βIII is first detectable at stages 12–13. Thereafter, the relative abundance of βIII in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non‐neural tissue extracts from older embryos (days 10–14) and hatchlings. Western blots of protein separated by two‐dimensional gel electrophoresis (2D–PAGE) reveal that the number of βIII isoforms increases from one to three during neural development. This evidence indicates that βIII is a substrate for developmentally regulated, multiple‐site posttranslational modification. Immunocytochemical studies reveal that while cβ₄ expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non‐proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that cβ₄ expression occurs either during or immediately following terminal mitosis, and suggests that βIII may have a unique role during early neuronal differentiation and neurite outgrowth.