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A nucleotidase present in rat liver cytosol which hydrolyzes certain 3′- and 5′-nucleotides, is subject to allesteric activation by dIno, dGuo, dThd, dUrd, and the corresponding 5′-deoxyribonuelcotides. A systematic study of enzyme activation as a function of effector concentration was carried out with Urd-3′-P or [14C]dThd-3′-P as substrate. It was found that optimal effector concentration was 6—8 mM for the nucleosides, 4 nm for dThd-5′-P, and 1—2 mM for the other 5′-deoxyribonucleotides. Deoxyinosine was a stronger activator than any of the other effectors and afforded a 3.3-fold increase in the rate of Urd-3′-P hydrolysis at optimal concentration. At 1 mM concentration the enzyme was stimulated 2.3 times by dIno and, furthermore, by dGuo dGuo-5′-P, dThd-5′-P, dUrd-5′-P, and dIno-5′-P in decreasing order of effectiveness. The di- and triphosphates of dThd were more stimulatory than the monophosphate at concentrations above 3 mM. The p-nitrophenyl ester of dThd-5′-P showed stimulation similar to that of the unesterified compound. A kinetic enzyme model was developed which explains all the observed relations between enzyme activity and effector concentration. The parameters of the model were calculated by means of a programmed electronic computer and showed that the apparent dissociation constants for binding of dIno and the 5′-monophasphates of dIno, dGuo, and dUrd to the regulatory site were about 1.7 mM. The other effectors had higher dissociation constants. The apparent Km values of the 5′-deoxyribonucleotides, calculated by means of the computer, were about 0.2 mM. This value is similar to the Km values of the 3′-nucleotides. The stimulation of enzyme activity is due to an increase in F without a detectable change in the apparent Km, a mechanism which is less common for regulatory enzymes. [ABSTRACT FROM AUTHOR]