Analyses of split ejaculates and urine voided before and after prostatic massage have been used to evaluate the origin of beta₂-microglobulin (b₂m) and carcinoembryonic antigen (CEA) in human semen. CEA and b₂m were present in rather high concentrations in all the fractions of the split ejaculates. The urine voided after massage of the prostate also contained increased levels of CEA and beta₂-microglobulin. Split ejaculates, selected from the electrophoretic pattern obtained for each fraction, revealed that b₂m in seminal plasma originates from the prostate and seminal vesicles in equal amounts. Besides the “free” b₂m in seminal plasma, human semen contains a sperm-bound b₂m fraction. Carcinoembryonic antigen was found in the highest concentrations in the second fraction of split ejaculates indicating that it originates mainly from the prostate.