Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.