Relaxin is a polypeptide hormone that activates the leucine-rich repeat containing G protein-coupled receptors, LGR7 and LGR8. In an earlier study, we reported that relaxin produces a biphasic time course and the second wave of cAMP is highly sensitive to phosphoinositide-3 kinase inhibitors (LY294002 and wortmannin). LY294002 inhibits relaxin-mediated increases in cAMP production by 40-50% across a large range of relaxin concentrations. Here we show that protein kinase C zeta (PKCzeta) is a component of relaxin signaling in THP-1 cells. Sphingomyelinase increases cAMP production due to the release of ceramide, a direct activator of PKCzeta. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin induced cAMP production to the same degree (approximately 40%) as LY294002. Relaxin stimulates PKCzeta translocation to the plasma membrane in THP-1, MCF-7, pregnant human myometrial 1-31, and mouse mesangial cells, as shown by immunocytochemistry. PKCzeta translocation is phosphoinositide-3 kinase dependent and independent of cAMP production. Antisense PKCzeta oligodeoxynucleotides (PKCzeta-ODNs) deplete both PKCzeta transcript and protein levels in THP-1 cells. PKCzeta-ODNs abolish relaxin-mediated PKCzeta translocation and inhibit relaxin stimulation of cAMP by 40%, as compared with mock and random ODN controls. Treatment with LY294002 in the presence of PKCzeta-ODNs results in little further inhibition. In summary, we present a novel role for PKCzeta in relaxin-mediated stimulation of cAMP.