Antagonists of growth hormone-releasing hormone (GHRH) inhibit growth of various human cancers including breast cancer, xenografted into nude mice or cultured in vitro. Splice variants (SVs) of receptors for GHRH have been found in several human cancers and cancer cell lines. The antiproliferative actions of GHRH antagonists could be mediated in part through these SVs of GHRH receptors. In this study we examined the expression of mRNA for GHRH and SVs of its receptors in human breast cancer cell lines MCF-7, MCF-7MIII, MDA-MB-231, MDA-MB-435, MDA-MB-468, and T47D. mRNA for GHRH was present in all lines tested. mRNA for SV1 isoform of GHRH receptors was found in MCF-7MIII, MDA-MB-468, and T47D; and for SV2 isoform in MCF-7MIII and T47D cell lines. In proliferation studies in vitro, the growth of T47D cells was stimulated by GHRH and dose-dependently inhibited by GHRH antagonist JV-1-38. H89 (protein kinase A inhibitor), bisindolylmaleimide I (protein kinase C [PKC] inhibitor) and verapamil (voltage-dependent calcium channel blocker) inhibited the GHRH-stimulated proliferation of T47D cells. The GHRH antagonist JV-1-38 suppressed the T47D cell growth in vitro stimulated by PKC activator (phorbol-12-myristate-13-acetate). The stimulation of T47D cells by GHRH was followed by an increase in cAMP production and GHRH antagonist JV-1-38 competitively inhibited this effect. Our results suggest that SVs of GHRH receptors could mediate the responses to GHRH and GHRH antagonists in breast cancer through Ca2+-, cAMP- and PKC-dependent mechanisms. The presence of SV1 of GHRH receptors in human cancers provides a rationale for antitumor therapy based on the blockade of this receptor by specific GHRH antagonists.