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Whole blood (WB) methods requiring less than 4 ml heparinized peripheral blood were used to define mononuclear cell phenotype, lymphocyte proliferation, and natural cell-mediated cytotoxicity (CYT) in samples from normal controls and patients with immunodeficiency states of acquired immunodeficiency syndrome (AIDS) and AIDS-related complexes (ARC). Results from two-color direct immunofluorescence staining of blood samples with monoclonal antibodies (Mabs) conjugated to phycoerythrin (PE) or to fluorescein isothiocyanate (FITC) and flow cytometry for the determination of mononuclear cells reactive with T11, T4, T8, B1, and NKH.1 surface markers were compared to one-color indirect immunofluorescence analysis with unlabeled Mabs and FITC-labeled goat anti-mouse IgG and flow cytometry. We found that two-color analysis was as sensitive as one-color analysis in detecting abnormal subset distribution in the patient groups. Functional properties of mononuclear cells (MNC) in WB samples and after density gradient separation were studied by mitogen-induced lymphocyte proliferation and CYT. Although the means of the groups studied varied depending on method used, results using WB methods clearly delineated the expected differences between the immunodepressed patients and normal subjects. The effects of sample storage on results obtained with WB methods for surface marker analysis, lymphocyte proliferation, and natural killer activity were also examined.