The aim of this study was to investigate putative effects of calcitonin gene-related peptide on developing dopaminergic neurons in the ventral mesencephalon. To determine a time-point for a physiological role of calcitonin gene-related peptide in the development of this system, we first investigated calcitonin gene-related peptide messenger RNA expression in the ventral mesencephalon of Wistar rats at embryonic days (E) 11-19. Calcitonin gene-related peptide messenger RNA was not detectable at E11, i.e. prior to the appearance of dopaminergic neurons in this area. From E14 to E19, calcitonin gene-related peptide messenger RNA was expressed in increasing amounts. We therefore investigated the effects of calcitonin gene-related peptide on serum-free cell cultures established from the E14 midbrain floor. Addition of calcitonin gene-related peptide (200 ng/ml) every other day significantly increased neuronal differentiation, including longer tyrosine hydroxylase-positive neurites, enhanced immunoreactivity for growth-associated protein-43 and increased dopaminergic uptake per neuron. These effects were maximal after seven to eight days. Calcitonin gene-related peptide acted synergistically with fibroblast growth factor-2 on these parameters. In contrast to fibroblast growth factor-2, however, calcitonin gene-related peptide did not promote survival of tyrosine hydroxylase-immunoreactive neurons. Lack of calcitonin gene-related peptide expression in the mesencephalon at E11 was paralleled by a lack of effect of calcitonin gene-related peptide on early presumptive dopaminergic neurons in terms of eliciting this phenotype.Our data suggest that calcitonin gene-related peptide may act physiologically as a differentiation-promoting factor for phenotypically defined dopaminergic neurons during a time period when dopaminergic neurons assemble in the ventral mesencephalon and grow axons towards their targets.