Cryopreservation is a method commonly used to store human blood samples. We sought to determine if cryopreserved peripheral blood mononuclear cells (PBMC) could be separated effectively into distinct populations by using monoclonal antibodies and immunomagnetic microspheres. PBMC obtained from healthy blood donors and from human immunodeficiency virus-infected subjects were cryopreserved for as long as 18 months. Recovered cells were separated into CD14+ monocytes and CD4+ T-cell subsets by immunomagnetic selection. Flow cytometry analysis indicated >95% depletion of monocytes from PBMC following immunomagnetic selection with anti-CD14. A highly enriched population of CD4+ T cells was obtained from the CD14-depleted cell fraction by using an anti-CD4 monoclonal antibody and detachable immunomagnetic beads. The CD4+ T cells were subsequently separated into CD4+ CD45RO and CD4+ CD45RA fractions. Each fraction contained >90% enrichment for the respective subpopulation and <5% of the reciprocal subpopulation. No significant differences in cell surface expression of leukocyte markers, in efficiency of selection of PBMC subpopulations, or in mitogen-induced proliferation were detected in freshly isolated or cryopreserved cells. Efficient recovery of cryopreserved specimens means that targeted assays can be performed on selected, prospectively stored samples once clinical endpoints have been achieved.