To alter neural networks in a restricted area of the brain, we previously developed the hydro-polymer gel (PG) implantation technique. In this study, we found that bilateral or unilateral injection of PG (1.6 microliters) into the ventromedial nucleus of the hypothalamus (VMH), contrary to injections into the preoptic area (POA) or posterior hypothalamic area (PHA), severely attenuated circadian changes of locomotor activity in blinded female rats. In addition, PG injection into the VMH also suppressed circadian changes in serum melatonin levels and induced persistent estrus. The effect of PG injection into the VMH on free-running rhythm was blocked by a complete cut around the VMH or a dorsal cut of the VMH but not by anterior or posterior cuts of the VMH. These results suggest that PG injection into the VMH induces some form of neural output via a dorsal route of the VMH that affects the generation of circadian activity rhythm.