GnRH and its receptor are expressed in human reproductive tract cancers and direct antiproliferative effects of GnRH analogs have been demonstrated in cancer cell lines. The intracellular signalling responsible for this effect differs from that mediating pituitary gonadotropin secretion. The GnRH structure-activity relationship (SAR) is different for the two effects. Here we report a SAR study of GnRH agonist antiproliferative action in model cell systems of rat and human GnRH receptors stably-expressed in HEK 293 cells. GnRH II was more potent than GnRH I in inhibiting cell growth in the cell lines. In contrast, GnRH I was more potent than GnRH II in stimulating inositol phosphate production; the signalling pathway in gonadotropes. The different residues in GnRH II (His⁵, Trp⁷, Tyr⁸) were introduced singly or in pairs into GnRH I. Tyr⁵ replacement by His⁵ produced the highest increase in the antiproliferative potency of GnRH I). Tyr⁸ substitution of Arg⁸ produced the most selective analog, with very poor inositol phosphate generation but high antiproliferative potency. In nude mice bearing tumors of the HEK 293 cell line, GnRH II and an antagonist administration was ineffective in inhibiting tumor growth but D-amino acid stabilized analogs (D-Lys⁶ and D-Arg⁶) ablated tumor growth. Docking of GnRH I and GnRH II to the human GnRH receptor molecular model revealed that Arg⁸ of GnRH I makes contact with Asp³⁰², while Tyr⁸ of GnRH II appears to make different contacts suggesting these residues stabilise different receptor conformations mediating differential intracellular signalling and effects on gonadotropin and cell growth. These findings provide the basis for the development of selective GnRH analog cancer therapeutics which directly target tumor cells or inhibit pituitary gonadotropins, or do both.