Recent studies strongly suggest that functional olfactory receptor cell innervation is necessary for the maintenance of the dopamine phenotype in the adult rat olfactory bulb. To determine whether afferent innervation is required for the initial expression of the dopaminergic phenotype during development, the current studies investigated the association between afferent innervation and phenotypic expression using both in vivo and in vitro systems. Ontogeny of the dopamine phenotype in the rat main olfactory bulb was assessed by the appearance of immunoreactivity for tyrosine hydroxylase, the first enzyme in the dopamine biosynthetic pathway. Development of receptor afferent innervation of the bulb was demonstrated with olfactory marker protein immunoreactivity. Tyrosine hydroxylase-immunoreactive cells occurred only in regions of the olfactory bulb receiving afferent innervation. However, the appearance of afferent fibers in the olfactory bulb preceded tyrosine hydroxylase expression by three to four days (gestational days 14-15 versus 18, respectively). In explant cultures, significant numbers of tyrosine hydroxylase-containing cells were observed only in en bloc co-cultures of presumptive olfactory bulb and epithelium. Explant cultures of presumptive olfactory bulb alone contained few, if any, tyrosine hydroxylase-immunoreactive cells. Similarly, explants produced by recombining previously separated presumptive olfactory bulb and epithelium exhibited very few tyrosine hydroxylase-immunostained cells. These data suggest that expression of the dopamine phenotype, as indicated by the presence of tyrosine hydroxylase, depends on a critical level of afferent innervation. The results also support previous studies which indicated that neuronal activity or an activity-dependent process may be required for induction of tyrosine hydroxylase expression.