Guanosine–inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin ( Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80 kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca 2+ appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2 mM exogenous CaCl 2 or Ca(NO 3) 2, with K a = 0.5 mM (estimated for CaCl 2). The K m values estimated for guanosine and inosine were 2.7 ± 0.3 μM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2′-Deoxyguanosine, 2′-deoxyinosine, 2′-methylguanosine, pyrimidine nucleosides and 5′-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1- N 2-ethenoguanosine and 1- N 2-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl 2 or ZnCl 2 inhibited the hydrolysis of guanosine with I 50 ≈ 60 μM. Whereas 2′-deoxyguanosine, 2′-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction ( K i values were 1.5, 3.6, 21 and 9.7 μM, respectively), hypoxanthine was a weaker inhibitor ( K i = 64 μM). Adenine, ribose, 2-deoxyribose, 5′-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine–inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.