Effects of neonatal maternal separation (MS) on global DNA methylation (5-methylcytosine) in the adult Wistar Kyoto (WKY) rat brain. WKY rats were exposed to daily 180-min MS or 15-min neonatal handling (NH) between postnatal days (P) 1–14. Adult offspring were sacrificed and several brain regions were dissected: the hippocampus, paraventricular nucleus (PVN) of the hypothalamus, amygdala, septum, medial prefrontal cortex (mPFC), and bed nucleus of the stria terminalis (BNST). DNA was extracted and used to measure 5-methylcytosine, an indicator of global DNA methylation. MS exposure dramatically increased 5-methylcytosine levels in the adult WKY hippocampus relative to NH controls, but did not significantly alter DNA methylation levels in the PVN, mPFC, amygdala, septum or BNST Data represent mean ± SEM. **** Statistically significant differences at p < 0.0001.