The utility of CD4 lymphocytes in monitoring disease progression and prognosis of human immunodeficiency virus (HIV)-infected patients is well established. We have modified a previously described antibody cocktail to provide complete lymphocyte subset analysis on 100-200-microL samples of whole blood. This method optimizes accuracy of CD4 lymphocyte assessments and provides simultaneous assessment of four other lymphocyte subtypes of interest in specimens with absolute lymphocyte counts as low as 300 X 10(6)/L. Lymphocytes are classified as Thelper (CD3+CD4+); Tsuppressor (CD3+CD8+); Tnull (CD3+CD4-CD8-, putative gamma delta T-cell receptor); B (CD19+CD20+); or natural killer (CD3-CD16+CD56+). The method positively discriminates against contamination of lymphocyte scatter gates by monocytes and unlysed erythrocytes and is compatible with a variety of cell preparation procedures. Increased accuracy of CD4 lymphocyte determinations and simultaneous identification of other lymphocyte subsets whose relationship to disease progression is under study make this an efficient and informative method for disease monitoring and evaluation of therapy in HIV-infected patients.