Anemia, the most common hematological disorder in human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS), is associated with decreased quality of life and survival. Hypogonadism is prevalent in advanced HIV disease, however, low testosterone levels have not been customarily implicated in HIV-associated anemia. This study was undertaken to determine whether there is a relationship between testosterone levels and androgen use with anemia in HIV, and to characterize other clinical correlates of HIV-associated anemia. This was a cross-sectional, observational study of 200 HIV-positive patients at a public hospital HIV clinic from July 2000 to August 2001. A written questionnaire detailed previous and current medication use, opportunistic infections, and malignancies. Hematological and virological parameters, testosterone, and erythropoietin levels were measured; CD4(+) T lymphocyte count and viral load nadir and peak levels were obtained from the computerized medical record. Anemia was defined as hemoglobin <13.5 g/dl in men and <11.6 g/dl in women. Twenty-four percent of women and 28% of men were anemic. Anemia was associated with lymphopenia (adjusted OR 4.0, 95% CI 1.36-11.80), high erythropoietin levels (adjusted OR 7.73, 95% CI 2.92-20.48), and low testosterone levels (adjusted OR 3.27, 95% CI 1.01-10.60). Anemia was negatively associated with female sex (adjusted OR 0.30, 95% CI 0.11-0.85), current antiretroviral therapy (adjusted OR 0.43, 95% CI 0.20-0.95), current androgen use (adjusted OR 0.20, 95% CI 0.05-0.84), and macrocytosis (adjusted OR 0.23, 95% CI 0.09-0.61). Low testosterone levels may have a positive association and supplemental androgens a negative association with anemia in HIV disease.