Three basic arginine amidases with different affinities to lima bean trypsin inhibitor (LBTI) and aprotinin affinity columns were separated in the middle molecular weight (MMW) preparation obtained from Cellulofine GCL-2000 gel filtration of CM-cellulose adsorbed human seminal plasma and were tentatively called basic human seminal plasma arginine amidase-L (BHSAA-L, with affinity to LBTI), -A (BHSAA-A, with affinity to aprotinin), and -TH (BHSAA-TH, without affinity to either). Some enzymatic properties were measured, including Ki values of LBTI and human seminal plasma proteinase inhibitor (HSP-PI) toward present enzymes. The Ki values of LBTI toward BHSAA-L and -TH were lower than those of HSP-PI and no Ki values for LBTI toward BHSAA-L were observed. The Km values of BHSAA-L and -A to some tripeptidyl-p-nitroanilide substrates seemed relatively lower than that of BHSAA-TH.