Surgical castration (also known as orchidectomy, ORX) has been frequently performed to avoid uncontrolled breeding. However, it has some serious disadvantages. Several laboratories have developed chemical castration methods, using bilateral intratesticular injection (BITI) of simple chemical solutions. The present study was undertaken to compare the effects of ORX and of hypertonic saline BITI on the androgen-sensitive tissues such as pituitary and hypothalamus. Serum testosterone (T) levels of ORX animals and hypertonic saline BITI animals (SAL) after 4 weeks of the manipulations exhibited significantly drops as compared with the levels of intact animals (Intact:ORX:SAL = 7.74± 1.31:1.34±0.19:1.28±0.18 ng/ml, p<0.001). Both ORX and BITI method induced similar stimulatory effects on the pituitary gonadotropin subunits and hypothalamic KiSS-1 gene expressions. In contrast, the effects of ORX and hypertonic saline BITI on hypothalamic GnRH gene expression were different from these gene expressions, shown an inverse relationship between the two groups (Intact:ORX:SAL = 1:0.45±0.06:1:2.07±0.41:1.51±0.37 AU; ORX, p<0.001; SAL, p< 0.05). In conclusion, we provided evidence that hypertonic saline BITI method has equivalent efficacy of T depletion to surgical castration in rats. The present study suggests the hypertonic saline BITI could be a promising substitute to conventional surgical castration.