BACKGROUND Recent data indicate the implication of an IGF/IGFBP/protease system in the development of ectopic implants of endometrium (endometriosis) in the pelvic cavity. MATERIAL AND METHODS We characterized mitogens and protease(s) against IGFBP-3 in the peritoneal fluid of women with or without endometriosis using preparative isoelectric focusing (IEC) Proteineous fractions were tested for a) mitogenic activity on NIH/3T3 cells and KLE endometrial-like cells, b) proteinolytic activity against radiolabeled IGFBP-3, and c) protein analysis using SDS-PAGE. RESULTS A significant increase in the DNA content of cultured cell lines was found in PF fractions having slightly acidic or alkaline pH and containing mainly proteins with molecular weights between 50 and 25 kD. These fractions were also found to cleave IGFBP-3 into 25, 18 and 14 kD fragments. Both activities were present in the PF of subjects with or without endometriosis. CONCLUSION The preparative isoelectric focusing system would be an advantageous approach to investigate the possible implication of the PF proteases with IGFBP-3 limited proteolysis generating PF mitogen(s) which possibly participate in the pathophysiology of endometriosis.