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Cervical cytobrush sampling is a relatively non‐invasive method for obtaining mucosal cells from the female genital tract. To define mucosal immune cells sampled by cervical cytobrushing and to validate this approach for local immunity studies, we investigated the impact of human immunodeficiency virus (HIV) status and inflammation on the yield and composition of cervical cytobrush specimens. Cervical cytobrush samples were obtained from 89 chronically HIV‐infected and 46 HIV‐negative women. The HIV‐infected women had significantly higher yields of CD3⁺, CD45⁺, CD19⁺, CD14⁺, Langerin⁺ and CD24⁺ cells than the uninfected women. While cytobrush‐derived T cells from uninfected women were predominantly CD4⁺ (4·2 CD4 : 1 CD8), CD8⁺ T cells were predominant in HIV‐infected women (0·6 CD4 : 1 CD8). The majority of CD4⁺ and CD8⁺ T cells from HIV‐infected and uninfected women were …