BACKGROUND Basophils are increasingly utilized as indicators of allergic inflammation and as primary allergic effector cells to study signalling pathways. However, until the present, their enrichment has been time consuming, costly and limited to relatively few specialized laboratories. OBJECTIVE We have therefore devised a reproducible and rapid method for the purification of human basophils from small quantities of peripheral blood within 1.5 h, which does not require the use of specialized equipment such as elutriators. METHODS Human basophils were obtained from healthy volunteers undergoing venipuncture. Heparinized or K3-ethylenediaminetetraacetic acid blood samples were first subjected to centrifugation in Hetasep, directly followed by negative selection using immunomagnetic beads. Basophil morphology and purity were assessed by May-Grünwald staining of cytospins. IgE-mediated histamine release was analysed spectrofluorometrically and IL-4 and IL-13 production by quantitative RT-PCR. CD203c and CD63 surface expression was measured using flow cytometry before and after activation with anti-IgE. RESULTS Using this protocol, basophils were enriched close to homogeneity in most cases with a mean purity of 99.34+/-0.88% (range 97-100%, n=18) and a mean recovery of 75.6 (range 39-100%, n=8). Basophil viability following purification was 99.6+/-0.89% using Trypan blue exclusion. The purification procedure gave rise to basophils with normal functional responses to anti-IgE regarding histamine release as well as IL-4 and IL-13 mRNA expression. Moreover, constitutive cell-surface CD203c/CD63 expressions were not elevated before anti-IgE stimulation. CONCLUSIONS The rapidity, simplicity and reproducibility of this method will facilitate the employment of basophils in high-output ex vivo studies.