AbstractCirculating immature blood cells are inhomogeneous in most cytometric parameters, including CD34 expression. This surface antigen forms patches, and we studied their spatial distribution pattern by processing staggered and digitalized microscopical images; the number, fluorescence intensity, and volume were determined in randomly identified CD34‐positive cord blood cells in suspension. Quantitative fluorescence analysis of individual CD34‐labeled cells was performed after acquisition of microscopical images in high resolution by using a CCD camera and adjacent deconvolution. Two major (n = 42 and n = 41) and one minor (n = 5) group of haematopoietic progenitors with different CD34+ distribution patterns were detected. All CD34 antigen patches are localized on the spherical cell membrane, and differ most significantly in fluorescence intensity (P < 10−4), antigen volume (P < 0.004), and patch number per cell (P < 0.02). If all parameters are employed, 96.6% of CD34+ cells are correctly classified into one of the three groups as shown by discriminant analysis. If fluorescence intensity is eliminated, recognition efficiency decreases to 68.2%, indicating that this is the most powerful single parameter. While total fluorescence intensity is most characteristic for each group, total patch volume and number per cell, and heterogeneity of patch volumes and their spatial distribution also contribute significantly to reliable classification into their groups. © 2007 International Society for Analytical Cytology