Objective: To compare the efficacy of two density gradient centrifugation media for retrieving spermatozoa from semen samples by evaluating the total motile sperm count (TMSC) and the percentage recovery.Methods: Twenty-two men with different sperm counts participated in the study. The samples were divided into two equal aliquots and processed using the commercial ISolate Sperm Separation Medium (Irvine Scientific, United States) and the GV Gradiente (IngáMed, Brazil). After separation, samples were counted and evaluated for motile sperm recovery.Results: The mean TMSC in the fresh sample was 19.65±21.08 million/mL. After the ISolate separation the TMSC was 6.71±7.29 million/mL, and for the GV Gradiente it was 6.27±6.82 million/mL. The percentage of motile spermatozoa recovered was 36.47%±21.61 for ISolate and 35.22%±21.24 for the GV Gradiente (p>0.05). The samples from 6 oligospermic patients (27%) were evaluated separately and the TMSC for ISolate was 4.83±2.92 million/mL, and for the GV Gradiente, it was 4.16±3.12 million/mL (p=0.54). When evaluating only normospermic patient samples, the TMSC for ISolate was 9.05±7.29 million/mL, and for the GV Gradiente, it was 8.47±6.79 million/mL (p=0.83).Conclusions: There was no statistical difference in retrieving motile sperm using the GV Gradiente and the ISolate Separation Medium.