Dialyzed rabbit liver cytosol was specifically freed of endogenous fructose-1,6-diphosphatase by immunoadsorption on a column of Sepharose-immobilized anti-fructose-1,6-diphosphatase. This material increased the specific activity of homogeneous enzyme to the maximal rate observed with EDTA and shifted the pH optimum from 8.4 to 7.4. With oleate or other fatty acids as activators, the hydrolysis of fructose-1,6-diphosphatase by enzyme, at neutral pH, showed nonlinear initial rates dropping to lower linear rates. Cytosol activator acted synergistically with oleate both to increase neutral enzyme activity and to maintain the high initial catalytic rates. After sucrose density centrifugation or gel filtration, the cytosol had no effect by itself, but still potentiated oleate activation. The factor was destroyed by treatment with subtilisin or trypsin, but all attempts to identify a unique protein component in cytosol were unsuccessful. The presence of Na dodecyl-SOJ, deoxycholate, or urea did not improve the resolution of the factor, but these compounds did lower the K50 for activation by cytosol. Since fatty acids are the only unique compounds which have been isolated from cytosol which activated fructose-1,6-diphosphatase, it appears that soluble proteins can act as natural carriers for the fatty acids. This was supported by the fact that both dialyzed rabbit alpha-globulins and muscle phosphofructokinase also acted synergistically with oleate in a manner similar to cytosol. Phosphatidic acid and phosphatidylserine activated fructose-1,6-diphosphatase, and their action was synergistic with oleate. Glutathione (1 mM) activated the enzyme 5-fold at pH 7.3 and its effects were additive with oleate and cytosol or alpha-globulins.