We investigated whether the repulsive SLIT/ROBO pathway is expressed in the endometrium and is negatively regulated during implantation. We also examined whether deficient expression in the Fallopian tube (FT) may predispose to ectopic pregnancy (EP). Endometrium (n=21) and FT (n=17) were collected across the menstrual cycle from fertile women with regular cycles. FT (n=6) was obtained from women with EP and decidualised endometrium (n=6) was obtained from women undergoing termination. SLIT/ROBO expression was quantified by RT-PCR and protein localised by immunohistochemistry. The regulation of SLIT/ROBO expression in vitro, by sex steroids and hCG, was assessed in endometrial (hTERT-EEpC) epithelial cells, and the effects of Chlamydia trachomatis (CT) infection and smoking was studied in oviductal (OE-E6/E7) epithelial cells. Endometrial SLIT3 was highest in the mid-secretory phase (P=0.0003) and SLIT1,2 and ROBO1 showed a similar trend. ROBO2 was highest in proliferative phase (P=0.027) and ROBO3,4 showed a similar trend. SLIT2,3 and ROBO1,4 were lower in decidua compared to mid-secretory endometrium (P