Polyacrylamide gel electrophoresis was used to evaluate rete testis fluid (RTF) and plasma from the cauda epididymidis (CEP) obtained from conscious Holstein bulls. The major protein in RTF had an electrophoretic mobility similar to that of blood serum albumin, but the seemingly analogous protein in CEP had a slower relative mobility. Bovine CEP contains at least three proteins, one glycoprotein and possibly three other PAS-positive components not detected in RTF or blood serum. In addition, esterases, acid phosphatases and several β-glucuronidases have been detected in CEP which were absent in RTF. Agarose immunoelectrophoretic analyses confirmed the basic differences among the three fluids. Although RTF and CEP share certain proteins with blood serum, each fluid is biologically unique. Certain proteins detected in CEP but not in RTF or blood serum probably originated from the testicular spermatozoa while others may represent epididymal secretion.