The objective of this study was to investigate the role of cytokines in the peri-ovulatory follicular fluid (FF) during IVF cycles. FF from 112 women was obtained during oocyte retrieval for IVF. The concentrations of five cytokines [interleukin (IL)- 1beta, IL-6, IL-12, IL-13, and tumour necrosis factor alpha (TNFalpha )] were measured in FF and their concentrations compared among women who became pregnant and those who did not. Thirty-one endometriosis patients, 15 idiopathic infertility, 21 tubal factor infertility, 15 ovarian factor infertility, and 30 patients with male factor infertility were included. Interleukin-13 and TNFalpha were absent in the peri-ovulatory FF of all patients. Fifty-two patients achieved pregnancy, whereas 60 did not. Both pregnant and non-pregnant groups were comparable for age, parity, ovarian stimulation parameters, fertilization rates, and embryo freezing rates. Concentrations of FF IL-1beta were not significantly different between pregnant and non-pregnant cycles. Concentrations of FF IL-6 were significantly higher in pregnant compared with non-pregnant cycles (P = 0.0005). Concentrations of FF IL-12 were significantly lower in pregnant compared with non-pregnant cycles (P = 0.0002). Higher concentrations of IL-12 and lower concentrations of IL-6 in the peri-ovulatory FF are associated with a negative outcome in IVF treatment. Interleukins-12 and IL-6 are biological markers that appear to affect IVF outcome.