Background: HIV-2 infection is characterized by low plasma viraemia and slower progression to AIDS in comparison with HIV-1 infection. However, antiretroviral therapy in patients with HIV-2 is less effective and often fails to provide optimal CD4 recovery. Methods: We examined viral tropism in persons with HIV-2 infection enrolled in the HIV-2 Spanish cohort. Viral tropism was estimated based on V3 sequences obtained from plasma RNA and/or proviral DNA. Results: From a total of 279 individuals with HIV-2 infection recorded in the Spanish national register, 58 V3 sequences belonging to 42 individuals were evaluated. X4 viruses were recognized in 14 patients (33%). Patients with X4 viruses had lower median CD4+ cell counts than patients with R5 viruses [130 (17-210) versus 359 (180-470) cells/mm3; P=0.007]. This was true even considering only the subset of 19 patients on antiretroviral therapy [94 (16-147) versus 184 (43-368) cells/mm3; P=0.041]. In multivariate analysis, significant differences in CD4+ cell counts between patients with X4 and R5 viruses remained after adjusting for age, gender, antiretroviral therapy and viral load. Conclusions: The presence of X4-tropic viruses in HIV-2 infection is associated with low CD4+ cell counts, regardless of antiretroviral treatment. Along with CD4+ cell counts, viral tropism testing may assist decisions about when to initiate antiretroviral therapy in HIV-2-infected individuals. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.