Neuronal microtubules have unique stability properties achieved through developmental regulation at the expression and post-translational levels on tubulins and microtubule associated proteins. One of the most specialized tubulins specific for neurons is class-III β-tubulin (also known as β6-tubulin). Both the upregulation and the post-translational processing of class-III β-tubulin are believed to be essential throughout neuronal differentiation. The present investigation documents the temporal and spatial patterns of class-III β-tubulin expression throughout neurogenesis. For this study a novel polyclonal antiserum named U-β6, specific to unphosphorylated class-III β-tubulin has been developed, characterized and compared with its commercial homologue TuJ-1. Our experiments indicate that the two antibodies recognize different forms of class-III β-tubulin both in vitro and in vivo. Biochemical data revealed that U-β6 bound unphosphorylated soluble class-III β-tubulin specifically, while TuJ-1 recognized both the phosphorylated and unphosphorylated forms of the denatured protein. In vivo U-β6 was associated with neurogenesis and labelled newly committed CNS and PNS neuroblasts expressing neuroepithelial cytoskeletal (nestin and vimentin) and surface markers (the anti-ganglioside supernatant, A2B5 and the polysialic acid neural adhesion molecule, PSA-NCAM), as well as differentiating neurons. These studies with U-β6 illustrate three main developmental steps in the neuronal lineage: the commitment of neuroepithelial cells to the lineage (U-β6 +ve/TuJ-1 –ve cells); a differentiation stage (U-β6 +ve/TuJ-1 +ve cells); and, finally, neuronal maturation correlating with a drop in unphosphorylated class-III β-tubulin immunostaining levels. These investigations also conclude that U-β6 is an earlier marker than TuJ-1 for the neuronal lineage in vivo, and it is thus the earliest neuronal lineage marker known so far.