ABSTRACT INTRODUCTION Flow cytometry allows immunophenotypic characterization of important lymphocyte subpopulations for diagnosis of diseases such as cancer, autoimmune diseases, immunodeficiencies and some infections. Normal values of rare lymphoid cells in blood, quantified by cytometry, vary among different populations; so it is indispensable to obtain normal national values that can be used in clinical practice. OBJECTIVE Characterize distribution of rare T-lymphocyte populations in peripheral blood, specifically double-positive T, natural killer T and activated T lymphocytes, as well as their relationship to sex and age. METHODS A cross-sectional study was carried out in 129 adults (68 women, 61 men) aged >18 years, without chronic diseases or unhealthy habits, who signed informed consent. Peripheral blood was collected for immunophenotyping of lymphocyte subpopulations with monoclonal antibodies specific for CD4+CD8+ double-positive T cells, CD3+CD56+ natural killer T cells, and CD3+CD25+HLA-DR+ activated T cells. An eight-color flow cytometer (Beckman Coulter Gallios) was used. The analytic strategy was modified, associating variables of interest in a single graphic, using conventional monoclonal labeling antibodies. Medians and minimum and maximum percentiles (2.5 and 97.5, respectively) were used as descriptive statistics, stratified by sex, for cell counts and percentages. A linear regression model was applied to assess age effects and a two-tailed Mann-Whitney U test for independent samples was used to assess sex differences. The significance threshold was set as p ≤0.05. RESULTS Median percentages of total lymphocytes: natural killer T cells 6.3% (1.4%–23%) in men and 4.7% (0.8%–11.3%) in women (p = 0.003); activated T cells 1.0% (0.2%–2.2%) in men and 1.2% (0.4%–3.1%) in women, without statistical significance; and double positives 0.8% (0.1%–4.2%) in men and 0.9% (0.3–5.1) in women, also without statistical significance. Median cell counts (cells/μL) were: natural killer T cells, 126 (27–580) in men and 105 (20–279) in women (p = 0.023); activated T cells: 20 (4–46) in men and 25 (7–75) in women, (p = 0.013) and double-positive T cells: 17 (2–85) in men and 21 (7–154) in women, without statistical significance. Sex influenced natural killer T cells, but age did not. CONCLUSIONS Age does not affect counts and percentages of rare T lymphocyte subpopulations in the blood of healthy Cuban adults. Sex differences found for some phenotypes suggest the need for different reference values for women and men.