Neurotensin (NT) neurons in the rat preoptic area are implicated in female-specific regulation of reproduction. Estrogen markedly increases expression of mRNA encoding the neurotensin/neuromedin N (NT/N) precursor in several cell groups of the preoptic area, including the anteroventral periventricular nucleus, periventricular preoptic nucleus, and medial preoptic nucleus. In the present study, immunohistochemistry was performed on tissue from ovariectomized females with or without estradiol treatment to test the hypothesis that increased levels of NT accompany hormonal induction of NT/N mRNA in these cell group. Since colchicine treatment is required for visualization of NT-immunoreactive cell bodies, an additional objective of this study was to determine whether colchicine alters expression of NT/N mRNA in this area. Estradiol caused a pronounced increase in the number of NT-immunoreactive cell bodies in the anteroventral periventricular nucleus, as well as adjacent parts of the periventricular preoptic nucleus and medial preoptic nucleus. In the absence of colchicine, estradiol increased the number of NT-immunoreactive fibers in these same regions. Surprisingly, NT-immunoreactive cell bodies with intense staining were abundant in certain parts of the medial preoptic nucleus regardless of hormonal condition. NT-immunoreactive cell bodies were also numerous in certain regions where NT/N mRNA-expressing cells are scarce, and in two of these regions, the median preoptic nucleus and vascular organ of the lamina terminalis, estradiol substantially reduced the number of immunoreactive cell bodies. Treatment of ovariectomized females with colchicine induced expression of NT/N mRNA in the same regions where NT-immunoreactive cell bodies were unexpectedly numerous, thus providing a compelling explanation for the discordant distributions of the mRNA and peptide. Together with previous findings, the present results indicate that increased levels of NT accompany hormonal induction of NT/N mRNA in the anteroventral periventricular nucleus, as well as adjacent parts of the periventricular preoptic nucleus and medial preoptic nucleus. In other regions of the preoptic area, colchicine-inducible expression of NT/N mRNA confounds assessment of hormonal influences on NT synthesis. Multiple populations of neurons capable of NT synthesis can be distinguished in the rostral preoptic area on the basis of differential responsiveness to estrogen or colchicine, thereby providing additional evidence for functional heterogeneity among NT-synthesizing neurons in this region.