Background: The KISS1 protein suppresses metastasis of several tumor models without blocking orthotopic tumor growth, but the mechanism remains elusive. For its role in human sexual maturation, KISS1 protein is secreted and processed to kisspeptins, which bind to the G protein-coupled receptor GPR54. We tested the hypothesis that KISS1 secretion is required for metastasis suppression via GPR54. Methods: KISS1 containing an internal FLAG epitope with (KFM) or without (KFMΔSS) a signal sequence was transfected into C8161.9 human melanoma cells, which do not express endogenous KISS1. Whole-cell lysates and conditioned medium from C8161.9KFM and C8161.9KFMΔSS cells were collected and analyzed for kisspeptins by immunoprecipitation and enzyme-linked immunosorbent assay. GPR54 levels were measured using real-time reverse transcription-polymerase chain reaction. The ability of conditioned medium from C8161.9KFM and C8161.9KFMΔSS cells to stimulate calcium mobilization in GPR54-expressing Chinese hamster ovary cells (CHO-G) and in C8161.9 cells was evaluated. Metastasis was monitored in athymic mice (groups of 10 per experiment) that were injected with C8161.9KFM or C8161.9KFMΔSS cells labeled with enhanced green fluorescent protein. Survival of mice injected with C8161.9 or C8161.9KFM cells was analyzed by Kaplan-Meier methods. Results: Full-length KFM and KFMΔSS were detected in whole-cell lysates of C8161.9KFM and C8161.9KFMΔSS cells, respectively, but kisspeptins were detected only in conditioned medium of C8161.9KFM cells. In vivo, C8161.9KFM, but not C8161.9KFMΔSS, cells were suppressed for metastasis to lung, eye, kidney, and bone, with corresponding differences in mouse survival (median > 120 versus 42 days). C8161.9KFM cells seeded mouse lungs but did not form macroscopic metastases. Conditioned medium from C8161.9KFM, but not C8161.9KFMΔSS, cells stimulated calcium mobilization in CHO-G cells. GPR54 expression was low in C8161.9 cells, which were not stimulated by conditioned medium from C8161.9KFM cells. Conclusions: KISS1 secretion was required for multiple organ metastasis suppression and for maintenance of disseminated cells in a dormant state. The absence of GPR54 expression in C8161.9 cells (whose metastatic spread was suppressed by KFM) suggests that metastasis suppression is not mediated through this receptor. The results imply the existence of another KISS1 receptor and/or paracrine signaling. The findings raise the possibility that soluble KISS1, kisspeptins, or mimetics could be used to maintain tumor dormancy, rendering treatment of already disseminated tumor cells (i.e., micrometastases) a legitimate target. © The Author 2007. Published by Oxford University Press.