The human corpus luteum (CL) undergoes luteolysis, associated with marked tissue and vascular remodeling, unless conception occurs and the gland is "rescued" by human chorionic gonadotropin (hCG). In Drosophila the Slit gene product, a secreted glycoprotein, acts as a ligand for the roundabout (robo) transmembrane receptor. Together they influence the guidance and migration of neuronal and non-neuronal cells. In vertebrates three Slit (Slit1, Slit2, Slit3) and four Robo (Robo1, Robo2, Robo3/Rig-1, Robo4/Magic Robo) genes have been identified. ROBO1, SLIT2 and SLIT3 are also inactivated in human cancers and may regulate apoptosis and metastasis. Since processes such as apoptosis and tissue remodeling occur during the regression of the CL, the aim of this study was to investigate the expression, regulation and effects of the SLIT and ROBO genes in human luteal cells. Immunohistochemistry and RT-PCR revealed that SLIT2, SLIT3, ROBO1 and ROBO2 are expressed in luteal steroidogenic cells and fibroblast-like cells of the human CL. Furthermore, using real-time quantitative PCR, expression of SLIT2, SLIT3, and ROBO2 was maximal in the late-luteal phase and significantly reduced after luteal "rescue" in vivo with exogenous hCG (P<0.05). Additionally, hCG significantly inhibited SLIT2, SLIT3 and ROBO2 expression in cultured luteinized granulosa cells (P<0.05). Blocking SLIT-ROBO activity increased migration and significantly decreased levels of apoptosis in primary cultures of luteal cells (P<0.05). Overall these results suggest the SLIT/ROBO pathway could play an important role in luteolysis in women.