ABSTRACT INTRODUCTION Double-negative T lymphocytes act as immunomodulators in immune response. This subpopulation is rare in blood but important in the immunopathogenesis of autoimmune diseases, viral infections, cancer and transplant rejection. These disorders have been studied in Cuba using flow cytometry, but normal values of these cells have not yet been established. OBJECTIVE Estimate preliminary reference values for double-negative T lymphocytes according to sex and age in Cuban adults. METHODS A cross-sectional study was carried out in a population of 182 healthy adult residents of Havana: 93 women and 89 men aged 18–80 years with no chronic diseases, toxic habits (smoking, excessive alcohol or caffeine intake) or medications that might alter quantity or functioning of immune-system cells. Peripheral blood was drawn to determine immunophenotype with monoclonal antibodies. The phenotype of double-negative T lymphocytes was quantified as CD45+/CD3+/CD4-/CD8-/CD56- using a Gallios flow cytometer (Beckman-Coulter, France). Medians and ranges (to the 5th and 95th percentiles) were calculated for sex and age, for both percentages and absolute values. To evaluate the effects of sex and age, both variables as well as their interaction were included in a linear model. RESULTS Respective median and range values were total percentage values 3.4 (1.6–7.4) and total absolute values (cells/μL) 57.5 (23.0–157.0). The effect of age on lymphocyte values (percentage and absolute) was significant, with lower numbers in the 51–80 years' age group (p <0.001). Percentage values according to age group were: 18–25 years, 3.8 (2.2–7.4); 26–50 years, 3.7 (1.7–8.7); and 51–80 years 2.6 (1.3–6.6). Absolute values by age group were: 18–25 years, 90 (32.6–163.7); 26–50 years, 65 (28.8–184.0); and 51–80 years 38.5 (17.9–90.1). Desegregating data by sex and age: percentage of women aged 18–25 years 5.2 (2.1–7.8), 26–50 years 4.0 (1.8–7.7), and 51–80 years 2.5 (1.3–5.8); percentage of men aged 18–25 years 3.4 (2.3–7.3), 26–50 years 3.8 (1.5–8.7), and 51–80 years 2.6 (1.2–7.3). Absolute values: women aged 18–25 years 112.0 (40.0–153.1), 26–50 years 67.0 (26.7–138.3), and 51–80 years 40 (18.6–92.0); and men aged 18–25 years 71.5 (32.1–166.7), 26–50 years 61.5 (29.9–188.7), and 51–80 years 36 (13.5–81.7). The low sex*age interaction confirms these differences occur in both men and women. Values decrease with age, with a more abrupt fall starting at 50 years. CONCLUSIONS Estimated reference values were determined for absolute values and relative proportions of double-negative T lymphocytes in healthy Cuban adults according to sex and age. Age was found to have a significant effect.