OBJECTIVE: To analyze the acoustic reflex thresholds obtained in neonates with probe tones of 226 and 1000 Hz. Method: We studied 71 newborns, 36 females and 35 males, aged between 4 and 29 days, otoacoustic emissions, and absence of risk factors for hearing loss. All underwent reflexes using ipsilateral and contralateral probe tone of 226 and 1000 Hz. The ipsilateral acoustic reflexes were searched in 1000 and 2000 Hz stimuli and stimuli with contralateral acoustic reflexes at 500, 1000, 2000 and 4000Hz. The research was conducted at the Department of Speech Pathology, Nossa Senhora da Conceição Hospital of Porto Alegre, using the immittance Interacoustics Audiotest 425h. In the statistical analysis were applied t-Student test for paired samples and t-Student for independent samples. RESULTS: We observed a statistically significant difference (p <0.05) when comparing the probe tones of 226 and 1000 Hz and ipsilateral and contralateral acoustic reflexes to stimuli in 1000 and 2000 Hz in both ears. In comparing the probe tones between the ears find statistical significance for both stimulation with 2000Hz investigated in the contralateral reflex. We also observed a statistically significant comparison of the acoustic reflex thresholds in relation to gender in OD to the probe tone of 1000 Hz in all reflexes ipsilateral and contralateral stimuli presented with stimuli at 500 and 1000Hz. CONCLUSION: The analysis of the acoustic reflex thresholds found a statistically significant difference between the probe tones of 226 and 1000 Hz, gender and ear in neonates.