To gain insights into the molecular mechanisms that restrict the expression of the oxytocin gene to anatomically defined groups of neurons in the hypothalamus, we generated transgenic mice bearing bovine oxytocin genomic fragments. Appropriate neuron-specific and physiological regulation was observed in mice bearing transgene bOT3.5, which consists of the oxytocin structural gene flanked by 0.6 kilobase pair (kbp) of upstream and 1.9 kbp of downstream sequences. bOT3.5 is expressed in oxytocin magnocellular neurons in the mouse supraoptic nucleus and paraventricular nucleus, but transgene RNAs are excluded from vasopressin neurons. Replacement of the drinking diet of the transgenic mice with 2% (w/v) NaCl for 7 days significantly increased the abundance of bovine oxytocin transcripts in the supraoptic nucleus, but not in the paraventricular nucleus, in parallel with the endogenous mouse oxytocin RNA. Surprisingly, mimicry of the endogenous oxytocin gene expression pattern was lost with larger transgenes. Addition of 0.7 kbp of contiguous downstream sequences (transgene bOT) or linkage to the bovine vasopressin gene (transgene VP-B/bOT3.5) repressed hypothalamic expression. No mice were derived bearing transgene bOT6.4, which consists of the oxytocin structural gene flanked by 3 kbp of upstream and 2.6 kbp of downstream sequences, suggesting that the presence of this DNA is detrimental to normal embryonic development. These data suggest that while bOT3.5 contains sufficient cis-acting sequences to mediate expression to particular subsets of hypothalamic neurons, the overall regulation of the oxytocin gene is governed by multiple interacting enhancers and repressors.Ever since pioneering studies demonstrated that the posterior pituitary contains activities that stimulate uterine contraction (1) and milk ejection (2), the oxytocin (OT) 1 system has been a favorite model for the study of the regulation and function of an identified class of central peptidergic neurons. OT is synthesized as a prepropeptide in the cell bodies of anatomically defined groups (nuclei) of hypothalamic magnocellular neurons. This precursor is subject to cleavage and other modifications as it is transported down the axon to terminals located in the posterior pituitary (3). The mature peptide products (the nonapeptide OT and a putative carrier molecule termed neurophysin) are stored until neural inputs governed by physiological stimuli elicit their release into the general circulation (4). Through an interaction with OT receptors located in the uterus and mammary gland, OT stimulates smooth muscle contraction, leading to parturition or milk ejection.The single-copy structural gene encoding the OT prepropeptide consists of three exons and encompasses Ͻ1 kbp (5). The OT gene is highly homologous at both the structural and sequence level to the gene encoding the related neuropeptide vasopressin (VP). In mammals, the two genes are separated by a short intergenic region of 11 kbp in the rat (5) and of 3 kbp in the mouse (6) and are transcribed to...