This study compared CD4 and CD8 lymphocyte counts obtained by microvolume fluorimetry (MVF) with those derived by flow cytometry (FC). Samples from 192 patients with known or suspected HIV were analysed, and the distribution of CD4 counts for these samples ranged from 0 and 1,279/microl, with 142/192 (74%) of the samples having CD4 values of less than 400/microl. Good agreement between FC and MVF CD4 counts was found (MVF = 0.98 x FC + 7.30) although there was a minor constant inter-method bias of approximately +7 cells/microl for the MVF data. For CD8 counts there was a constant bias between the two methods of approximately +23 cells/microl for FC. Most outliers were associated with higher FC CD8 counts. Supplementary analyses showed a high level of agreement between FC and MVF methods for the CD4:CD8 ratios (MVF = 0.98 x FC). This suggests that observed discrepancies between FC and MVF methods were almost certainly a result of the influence of the absolute lymphocyte counts obtained from the haematology analyser. The results confirm that the IMAGN 2000 microvolume fluorimeter system can be used as an alternative to conventional flow cytometry for the enumeration of CD4 and CD8 counts.