Abstract. Objective: To determine genotoxicity, cytotoxicity and methemoglobinemia of a mixture of lidocaine (LD) and prilocaine (PL) (EMLA®) in an experimental model. Study design: Newborn hairless rats were studied in a prospective random and masked design. Groups I–III received topically LD-PL mixtures at 5, 10 or 20% respectively; group IV: petrolatum (negative control); group V: 5-ﬂuorouracil 5% (genotoxicity and cytotoxicity positive control); and group VI: aniline (methemoglobinemia positive control). Blood samples were collected every 24 h from 0–144 hours. Genotoxicity and cytotoxicity were assessed by the micronucleus assay. Changes in micronucleated erythrocytes and micronucleated polychromatic erythrocytes evaluated genotoxicity, and polychromatic erythrocytes in 1,000 total erythrocytes were used to determine cytotoxicity. Methemoglobin values were quantiﬁed by spectrophotometry. Results: The micronucleated erythrocytes number increased proportionally to the LD-PL mixture dose (p = 0.04), from 60% in group II until 90% in group III in relation to their basal value; however, no cytotoxicity effect was observed. Methemoglobin values increased in groups II, III, V and VI from the second day (p < 0.05).