The modification of the neural cell adhesion molecule (NCAM) with polysialic acid plays a pivotal role in the developing nervous system. Here we studied the expression and function of polysialic acid during development of the mesencephalic dopaminergic system of mice. Using immunohistochemistry, polysialic acid was detected on nestin-positive radial glia processes and on cell somata in the pial zone of the midbrain at embryonic day E11.5 and E14.5. As studied by quantitative real-time RT-PCR, mRNA profiles of NCAM and the polysialyltransferases, ST8SiaII and ST8SiaIV, matched the course of tyrosine hydroxylase, dopamine transporter, nur-related factor 1, and paired-like homeodomain transcription factor 3 expression, which were used as marker genes of dopaminergic development. Asking for a possible role of polysialylation during formation of the dopaminergic system, mice lacking polysialic acid because of ablation of both St8siaII and St8siaIV were analyzed at selected time points by tyrosine hydroxylase immunohistochemistry and by real-time RT-PCR of dopaminergic markers. Surprisingly, no differences between wild-type and mutant mice could be detected. Likewise, enzymatic removal of polysialic acid from cultured neurons of the ventral embryonic midbrain had no effect on the expression of dopaminergic marker genes. We conclude that despite its abundance polysialic acid is dispensable for the formation of the mesencephalic dopaminergic system.