Aims: Evaluate the effects of ayahuasca (AYA) on cocaine (COC)-induced conditioned place preference (CPP). Methods: (A) AYA was lyophilized, and its components were quantified by LC-MS/MS. (B) Six-week-old C57Bl/6 mice were habituated to the CPP apparatus for 15 minutes. The following day, they underwent a pre-test to assess their natural compartmental preference (15 minutes). During the conditioning phase (CT; 3rd-10th day), mice remained confined in one of the CPP compartments for 30 minutes. On the 11th day, they were tested to evaluate: (B.i) Effects of AYA on CPP: on odd CT days, they were conditioned with AYA (2.5; 7.5; 12.5 and 15mg DMT/kg v.o.), and on even CT days, with water. (B.ii) Competition between COC and AYA-induced CPP: on odd CT days, they were conditioned with COC, and on even CT days, with AYA (15 mg of DMT/kg). (B.iii) Acute administration of AYA on COC-CPP expression: mice were submitted to four COC CT sessions. On the test day, they received water or AYA (12.5 or 15mg of DMT/kg) (CEUA/FCF-019.2020-P608). Results: (A) AYA composition (mg/mL): DMT=1.38; Harmina=0.72; Harmaline=0.11; Tetrahydroharmine=0.84. (B.i) Only Aya15 induced CPP (p<0.001). (B.ii) COC-induced CPP prevailed over Aya15-CPP (p<0.01). (B.iii) Aya12 and 15 prevented COC-CPP (p<0.05). Conclusions: AYA has been a major focus of Brazilian studies on harm reduction measures triggered by drugs of abuse. This substance has a higher safety profile, low mortality rates, and low potential to induce substance use disorder. Our findings suggest that AYA may prevent COC-induced CPP expression in mice. Acknowledgment: FAPESP, CNPq.