Sympathetic ganglia contain 2 adrenergic derivatives of the neural crest: principal neurons and small, intensely fluorescent (SIF) cells. The developmental mechanisms responsible for the generation of these 2 cell classes in vivo are not well understood. To examine the possible developmental and lineage relationships between differentiating principal neurons and SIF cells, a fluorescence microscopic study utilizing antibodies against tyrosine hydroxylase (TH) and catecholamine histofluorescence has been combined with the ultrastructural examination of embryonic and postnatal rat superior cervical ganglia (SCG). On embryonic day 12.5, before neuroblasts had become postmitotic, the cells in the SCG possessed intense TH immunoreactivity and had weak to bright catecholamine histofluorescence, but no cells displayed the fine structure of mature SIF cells or neurons. At embryonic days 16.5 and 18.5, postmitotic principal neurons expressed more moderate levels of TH and catecholamines characteristic of the late embryonic and postnatal SCG. By contrast, a small number of cells containing intense TH or catecholamine fluorescence were present in embryonic day 16.5 and older ganglia. Almost all of the intensely fluorescent cells observed were found apposed to capillaries within the ganglion. These embryonic intensely fluorescent cells were larger than SIF cells seen postnatally. Ultrastructural examination of developing ganglia confirmed that cells containing numerous large, dense-cored vesicles (LDCVs) were a prominent feature of ganglia that also contained intensely fluorescent cells. In addition, some embryonic cells containing LDCVs were mitotic. From these and other studies, it seems likely that during development, neuron precursors, in response to differentiation factors such as fibroblast growth factor (FGF) and/or NGF, acquire overt neuronal traits and become postmitotic. Subsequently, cells resembling mature SIF cells appear next to blood vessels, where they may have received other instructional signals such as glucocorticoids. This developmental scheme suggests that the differentiation of principal neurons and SIF cells is independently regulated, and that the ability of SIF cells to convert into principal neurons observed in vitro cannot account for the generation of neurons in vivo.