An approach to perform lymphocyte subset analysis on frozen-thawed whole blood (F/T WB) is described. WB from 24 human immunodeficiency virus type 1 (HIV-1) seropositive individuals and 21 controls was analyzed fresh and after frozen storage (with or without dimethyl sulfoxide) at ุ80ยฐC, in liquid nitrogen (LN 2 ), and at ุ20ยฐC. Analysis of F/T WB utilized 3-color flow cytometry with CD45 and right angle light scatter gating. Absolute cell counts were obtained for 30 samples by using staining tubes containing internal bead standards [TruCount, Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA]. The mean difference between CD3 ุ 4 ุ percentages for F/T (ุ80ยฐC storage for up to 1 year) and fresh WB was less than ุ0.2% (95% limits ฯฎ3%, P โซุโฌ 0.5) with 39 of 45 (87%) results falling within 2% of the fresh values (P โซุโฌ 0.74). Absolute CD3 ุ 4 ุ cell counts for F/T WB were generally lower than corresponding results for fresh aliquots (median difference was 33 cells/ยตl, P ฯฝ 0.0001), but the results were highly correlated (r 2 โซุโฌ 0.975, P ฯฝ 0.0001). Results were more variable, although still highly correlated, for CD3 ุ 8 ุ cells, and with other freezing and storage conditions. It is concluded that lymphocyte subset analysis using F/T WB yields comparable results to fresh samples, which should prove useful for a number of practical applications.