Adenosylhomocysteinase from yellow lupin seeds (Lupinus luteus) has been purified to homogeneity. Active enzyme, Mr = 110000, consists of two probably identical subunits with Mr = 55000 as judged by gel filtration and dodecyl sulphage/polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was shown to be 4.9 +/- 0.1. It was demonstrated by disc and pore gradient electrophoresis that the most purified fraction formed multimers. The enzyme shows optimum activity at pH 8.5-9.0. Km values are 2.3 micrometer, 4.6 mM and 12 micrometer for adenosine, DL-homocysteine and S-adenosyl-L-homocysteine, respectively. The energy of activation for S-adenosylhomocysteine synthesis was estimated as 14.4 kcal/mol (60.2 kJ/mol) and temperature coefficient as 2.4. The equilibrium constant for the hydrolysis of S-adenosylhomocysteine amounts to 5 X 10(-7) M. Anti-sulfhydryl reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide acted as irreversible inhibitors. The enzyme exhibits high specificity for homocysteine whereas some of the rare nucleosides tested could substitute for adenosine.