Despite the fact that both peritoneal (PF) and follicular (FF) fluids have a common ovarian origin, FF is a natural inducer of sperm acrosome reaction (AR) while PF is not. To better understand these effects, concentrations of oestradiol, progesterone and proteins in peri-ovulatory PF and FF were determined and compared. PF was aspirated by laparoscopy at the peri-ovulatory stage from women with unexplained infertility. FF was collected from patients undergoing IVF and pooled. PF and FF were tested for the presence of antisperm antibodies. Oestradiol and progesterone were measured by enzyme immunoassay, and total protein concentration was determined and analysed. The AR was determined in spermatozoa that were exposed to PF alone, progesterone-supplemented PF, progesterone, control medium, or ethanol. No antisperm antibodies were found in any fluid tested. Oestradiol and progesterone and concentrations in PF were significantly lower than in FF. Protein concentration was also significantly lower in PF than in FF, but no differences were observed between the electrophoretic patterns. When capacitated spermatozoa were exposed to progesterone-supplemented PF there was a significant increase in the percentage of AR with respect to those in PF, control medium or ethanol. These results suggest that the lack of AR-stimulating activity of PF was related to its lower progesterone concentration compared with FF.