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The aim of the study was to analyze muscle activation in the three positions of the deadlift (DL). Twenty male participants (33.4 ± 3.9 years; 42.2 ± 9.1 months of experience with DL; 91.0 ± 14.8 kg; and 1.78 ± 0.06 m) pulled a bar through isometric actions in three DL positions: lift-off, mid-pull, and lockout. Isometric strength, knee angle, and activation of the rectus femoris (RF), biceps femoris (BF), lateral gastrocnemius (GAL), and erector spinae (ERE) muscles were collected. The analysis of variance showed that the maximum isometric force presented differences between the positions (p = 0.001; η2 = 0.973) considered large with higher values at the mid-pull position. Interactions were found between muscles and position (p = 0.001; η2 = 0.527) considered large. The RF and ERE showed greater activation in the lift-off position, while in the mid-pull position, there was greater activation of the BF and GAL muscles. The DL positions produce different activations in the bi-articular and uni-articular muscles. The lift-off requires more activation from the RF and ERE positions. The mid-pull position, despite generating greater force, presented greater activations in the BF and GAL. The ERE showed higher activations as the external torque was greater.