In this study of brushtail possums, proteins present in epididymal fluid and not present in blood plasma and those that become associated with spermatozoa as they pass along the tract were investigated. At least 19 proteins were present in epididymal fluid in the various regions of the tract that were not detected in serum. Some of these may be present on the sperm plasmalemma. Six proteins were extracted from caput spermatozoa (M(r) 117,000, 103,000, 87,500, 85,000, 62,000 and 33,000) that did not appear in the caudal sperm extracts. Eight proteins (M(r) 50,000, 49,000, 32,000, 27,000, 26,500, 25,000, 24,500 and 18,000) were localized to caudal sperm extracts. These findings suggest that some sperm proteins are lost or modified, whereas others are added to the sperm plasma membrane during epididymal transit. In vitro incorporation of [35S]methionine by the epididymal tissue showed that the distal caput and corpus are the most active regions in the synthesis and secretion of proteins. Four caudal epididymal proteins (M(r) 72,000, 31,000, 26,500 and 21,000) were partially sequenced. Those of M(r) 31,000 and 26,500 had the same N-terminal amino acid sequence suggesting post-translational modification of the same protein; they showed homology to a retinoic acid-binding protein. The protein of M(r) 72,000 was found to be homologous to alpha-fetoprotein, whereas the protein of M(r) 21,000 showed no significant homology to any protein in the database. These results show that the lumen of the epididymis has many proteins that are not present in the blood, some of which appear to become associated with spermatozoa during epididymal transit.