Flow cytometry analysis of lymphocyte subset markers was performed for a group of sexually active, human immunodeficiency virus (HIV)-negative adolescents over a 2-year period to establish normative data. Data were collected in the REACH Project (Reaching for Excellence in Adolescent Care and Health), a multicenter, longitudinal study of HIV-positive and high-risk HIV-negative adolescents. Two- and three-color flow cytometry data were collected every 6 months for these subjects. We determined the effects of gender, race, and age on the following lymphocyte subset markers: total CD4+ cells, CD4+ naïve cells, CD4+ memory cells, all CD8+ cells, CD8+ naïve cells, CD8+ memory cells, CD16+ natural killer cells, and CD19+ B cells. Gender was the demographic characteristic most frequently associated with differences in lymphocyte subset measures. Females had higher total CD4+ cell and CD4+ memory cells counts and lower CD16+ cell counts than males. Age was associated with higher CD4+ memory cell counts as well as higher CD8+ memory cell counts. For CD19+ cells, there was an interaction between age and gender, with males having significantly lower CD19+ cell counts with increasing age, whereas there was no age effect for females. Race and/or ethnicity was associated with differences in total CD8+ cell counts and CD8+ memory cell counts, although both of these associations involved an interaction with gender.