A hollow‐fibre immunoadsorption system has been developed for the purification of CD34+ cells from mononuclear cells. This cell separation technique is based on the use of uniform surface fluid shear stress to fractionate cells that attach to the inside surface of hollow fibres. Monoclonal antibody to the CD34 antigen was covalently coupled to the lumenal surface of cuprophan minidialysers (surface area 220 cm2). After the selective adsorption of CD34+ cells (28 min), a depleted fraction was collected at 5 dynes/cm2 followed by washes at 10 and 25 dynes/cm2. Antigen‐positive cells were recovered after incubation with chymopapain. The device was tested by using peripheral blood mononuclear cells from seven patients who had received granulocyte colony‐stimulating factor and chemotherapy. The average number of cells processed was 1.3 ± 0.2 × 108 (± S.E.M.), and the preselection incidence of CD34+ cells was 1.6 ± 0.6% (range 0.21–4.13%; n = 7). The enrichment purity was 94.4 ± 3.1%, and 61 ± 9% of input CD34+ cells were recovered in the enriched fraction (n = 4). Enrichment resulted in a 3.3 ± 0.1 log10 depletion of CD34− cells (n = 4). Hollow‐fibre affinity cell separation has potential as a medium to large‐scale cell enrichment technology.