In the female rat, behavioral and endocrine aspects of reproduction are controlled, in part, by the action of the steroid hormone estradiol on several regions of the brain, including the ventrolateral portion of the ventromedial hypothalamus (VL-VMN) and the arcuate nucleus of the hypothalamus (ARC). Quantitative assessment of the effects of estradiol on the regulation of ribosomal RNA in rat hypothalamus was accomplished in this study by tandem in situ hybridization experiments with 2 ribosomal DNA probes specific to the initial transcript (precursor) or mature, stable (product) rRNA. This novel approach allowed the regulation of RNA processing by steroid hormones to be analyzed in the individual neuron, a particularly important concern in heterogeneous tissue such as the brain. Estradiol was administered subcutaneously to ovariectomized rats for 15 min, 30 min, or 2 hr, or a discontinuous schedule of 2 hr on/7 hr off/2 hr on. Levels of precursor and product rRNA were measured in VL-VMN and ARC neurons using a computerized image-analysis system. Significant increases in the levels of precursor rRNA were observed only in the VL-VMN as early as 30 min after hormone exposure, with a doubling in the amount of precursor rRNA occurring at 2 hr. No changes in product rRNA were observed in either brain region at these early times. These data, in conjunction with our previous findings of increases in product rRNA after longer hormone exposure times, lead us to conclude that rRNA gene transcription is activated in rat hypothalamic neurons within 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)