Background: Nipple Aspirate Fluid (NAF) is constantly collected in the duct-lobular complex, and extracted by warming up and giving a massage for the breast with nipple aspiration.Its known that NAF is available from even healthy women without invasive procedure and it reflects the internal environment of duct-lobular unit and expected to be used for breast cancer risk evaluation.We tried sampling of NAF in the Japanese healthy women this time and analyzed it about the sampling rate with the affected factors and the characteristic of the protein composition using the surface enhanced laser disorption ionization time of flight mass spectrometry (SELDI-TOF-MS).Methods: We obtained the specimen according to the methods as follows after obtained participation consent in a document for a research that was approved by Komagome Hospital EC.NAF sampling method: 1)Warming of the breast with a heating pad and scrubbing the nipple to dislodge keratin plugs; 2) The Participants compressed her breast with both hands,and the plunger of the syringe was withdrawn to aspirate the nipple; 3) If a secreting fluid develops, we obtain a specimen with capillary.A method of the analysis by the SELDI-TOF-MS NAF dilutes 20 times in PBS and conducts protein analysis by Protein Chip System~.Results: We tried NAF sampling to 124 women from 21 to 65 years old from March 31, 2007 to July 25, 2009, and sampling was possible from 62 people (50%) (Sampling from the one side was possible in 41 people and both side in 21 people).NAF was classified roughly into white, transparence and serous macroscopically.The results about the protein profile analysis was: 1) Homogenous is high in the protein peak of NAF obtained from the right and the left of the same person; 2) When we compare white, colorless and serous NAF, there are few differences between the individual and forms a relatively homologous protein peak with the white specimen.Because the protein profiling was similar between white NAF and the milk, it was suggested that white NAF was a functional secretion.Conclusions: However NAF sampling rate was reported approximately 80-90% in the European and American report, the rate of Japanese women was 50%.The improvement of the sampling rate is expected in future by adding the appropriate choice of the study patients and the improvement of the sampling procedure.