We have previously shown that Ca²⁺ and its intracellular binding protein, calmodulin (CaM), are physiological modulators 6-phosphofructo-1-kinase (PFK), participating on the regulation of the enzyme activity by insulin. PFK is the principal regulator of the glycolytic flux, and so our data confirm the well documented material concerning the regulation of glycolysis by Ca²⁺ and CaM. Moreover, we have also shown that, clotrimazole (1-(a-2-chlorotrityl)imidazole, CLO), a widely used anti-mycotic agent which has been described as a CaM antagonist, decreases the glycolytic flux on human breast cancer cells detaching PFK from cytoskeleton, that is coupled to a decrease on cell viability. The aim of this study is to investigate the effects of CLO and other CaM antagonists, as compound 48/80 (C48/80), on the activity of purified PFK as well as its interaction with cytoskeketal elements in order to propose them as possible antitumoral agents. Our results shows that, in the presence of Ca²⁺, CLO but not C48/80, promoted a 50 % inhibition on purified PFK activity (10.0 + 0.7 and 5.1 + 0.9 mU/mg, for control and in the presence of 50 mM CLO, respectively, P < 0.01, Student’s t-test). On the other hand, in the presence of Ca²⁺ and CaM, as well as in the absence of both, CLO stimulated purified PFK activity 70 % (in the presence of Ca²⁺ and CaM) and 49 % (in the absence of the additives). C48/80 still did not altered purified PFK activity. These experiments were also performed using purified PFK in the presence of human erythrocytes membrane, where PFK binds becoming inhibited. Under these conditions, the pattern of CLO and C48/80 effects were overwhelmingly altered. In the presence of Ca²⁺, both CLO and C48/80 stimulated PFK activity 43 and 35 % respectively. This activation was similar to the one promoted by Ca²⁺ and CaM (39 %), which we have previously described to detach the enzyme from membrane. However, in the presence or in the absence of Ca²⁺ and CaM, CLO or C48/80 did not affect PFK activity measured in the presence of erythrocytes membrane. Altogether, our results support evidences that CLO, but not C48/80, directly modulate PFK activity, and that this modulation can be positive or negative depending on the presence or not of Ca²⁺ and CaM. In addition, both CLO and C48/80 affect interaction of PFK with erythrocytes membrane, modulating the enzyme activity.