The Pet-1 [pheochromocytoma 12 ETS (E26 transformation-specific)] gene plays a critical role in the development of serotonin (5-HT)-modulated behaviors via its control of embryonic 5-HT neuron differentiation. Pet-1 transcription is induced exclusively in 5-HT neuron postmitotic precursors before the appearance of transmitter, and its restricted expression is maintained in the adult. However, the mechanisms that direct Pet-1 expression to this single CNS neuronal cell type are unknown. Here, we show, using transgenic methods, that genomic sequences upstream, but not downstream or within the Pet-1-coding region, are sufficient for 5-HT neuron-specific transgene expression. Enhancer sequences within a 40 kb upstream fragment directed position-independent lacZ (β-d-galactosidase) transgene expression to the developing hindbrain before the appearance of 5-HT. Moreover, virtually all of the 5-HT neurons in the adult were lacZ positive in all of the lines examined. Transgene expression in 5-HT neurons was maintained when the 40 kb fragment was truncated on its 5′ end to either 12 or 1.8 kb, although position independence was then lost. Analysis of transgene expression in Pet-1 null mice indicated that Pet-1 was required to maintain the activity of the Pet-1 enhancer region in a subset of 5-HT neurons. These findings suggest that a conserved 1.8 kb region immediately flanking the Pet-1-coding region is a critical genomic target of the transcriptional cascade that governs 5-HT neuron development and provide additional evidence for 5-HT neuron heterogeneity at the genetic level. We discuss the potential application of the Pet-1 transgenes reported here to the selective genetic manipulation of 5-HT neurons.