The relative density of lymphocyte CD3, CD4, CD5, CD8, CD20, CD23, CD28, CD38, CD45RA, CD45RO, CD57 and HLA-DR antigens was measured as antibody binding capacity (ABC) in 58 blood donors aged 19-66 years. The group was analysed in order to obtain reference values (percentages and absolute numbers) for routine, quantitative three-colour flow cytometry (FC) tests, and we included around ten males and females for each of the 15 year age intervals. Whole blood was stained (30 min on ice) with FITC, PE or PerCP conjugated MAbs. The analysis was performed with a FACScan equipped with LYSYS II and Paint-a-GatePlus software. The instrument was calibrated daily with QC3, QuickCal (FITC and PE) and Calibrite and monthly with QSC and stained cells (which included also the control for PerCP performance). The ABC was measured with QSC (Flow Cytometry Standards Corporation). The CD4+ lymphocytes expressed significantly more CD3, CD28 and HLA-DR antigens, and less CD45RA antigen than the CD8+ cells (p < 0.0001). A significant decrease with age was observed for CD3 and CD45RA on both CD4+ and CD8+ subsets (p < 0.05). The lymphocytes of women, compared with those of men, showed decreased ABC for CD8, CD20 and CD28 antigens. The results illustrate the necessity for close matching of control with case groups. They also illustrate the possibilities of modern FC methods based on quantitative quality control and three-colour analysis.