Recurrent implantation failure leads to a reduced pregnancy rate. The expression patterns of trophinin and dipeptidyl peptidase IV (CD26) indicate the involvement of embryo implantation and early placental development. The purpose of the present study was to evaluate endometrial co-culture cells in the presence of embryo with trophinin and CD26 immunofluorescence staining. Patients with recurrent implantation failure were enrolled in the present study. The patients were aged between 26 and 36 years. Co-cultures were prepared from endometrial biopsies for each patient. Controlled ovarian hyperstimulation was performed on each of the patients. Certain embryos were maintained in a conventional culture environment (n=80), and others in an endometrial co-culture environment (n=25). Following embryo transfer, the co-culture cells were examined under an inverted wide-field fluorescence microscope. The ratio of a successful pregnancy was 0.38 in the present study (n=5/13 pregnancies). The average age of the successful group (28 +/- 3.54 years) was younger compared with the unsuccessful (32.67 +/- 2.81) group (P <= 0.05). The number of trophinin (+) endometrial cells in the presence of an embryo was significantly lower (P=0.046) in the successful group on the first day. No significant difference between the groups was observed in terms of the number of CD26 (+) cells on the first to the fourth days (P <= 0.05). Trophinin and CD26 immunostaining is important in the early period of pregnancy, and it will be beneficial in terms of providing the deficit of conventional culture medium in performed studies with the endometrial co-culture medium. The co-culture may be important, particularly in the early period, in patients with recurrent implantation failure in terms of enabling a connection between the cells belonging to the endometrium and the embryo.