Tumour necrosis factor-α (TNF-α) is a pleiotropic cytokine synthesized throughout the female reproductive tract. Even though evidence has accumulated that supports its role in autocrine and paracrine processes, its expression and function in the human endometrium are still not completely understood. To gain a better understanding of the synthesis and release of TNF-α in the endometrium and how this relates to concentrations in uterine secretion, its expression throughout the menstrual cycle was investigated by three different techniques. Samples of endometrial tissue and uterine secretions were collected from patients undergoing abdominal and vaginal hysterectomy for benign reasons. The mRNA expression of TNF-α was investigated in homogenized endometrial tissue by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) (n = 18). An assessment of the cellular TNF-α protein localization in the endometrial glands was performed immunohistochemically (n = 39). The concentrations of the secreted TNF-α protein in endometrial secretion were determined by enzyme-linked immunosorbent analysis (n = 30). All three methods gave similar results on the temporal expression of TNF-α mRNA and TNF-α protein during the cycle. Concentrations of endometrial TNF-α mRNA in tissue samples and TNF-α protein in uterine secretion were quite low at the beginning of the cycle, rose sharply in the mid- to late proliferative phase and decreased towards the end of the cycle. The concentrations of TNF-α protein in the endometrial glands, as shown by immunohistochemical investigation, stayed high throughout the secretory phase at values slightly below those of the late proliferative phase.