A biannual external quality assurance (EQA) scheme for flow cytometric typing of the HLA‐B27 antigen is operational in The Netherlands and Belgium since 1995. We report here on the results of the first seven send‐outs to which 36 to 47 laboratories participated. With the send‐out, four specimens from blood bank donors, who had been typed for HLA Class I antigens by complement‐dependent cytotoxicity, were distributed. Subtyping of the HLA‐B27 allele was performed by PCR‐SSP. Ten samples were HLA‐B27pos (all HLA‐B*2705) and 18 were HLA‐B27neg. For flow cytometry, the most widely monoclonal antibody (MoAb) used was FD705, followed by GS145.2 and ABC‐m3. The majority of laboratories used more than 1 anti‐HLA‐B27 MoAb for typing. The HLA‐B27pos samples were correctly classified as positive by the large majority of participants (median 95%; range 85% to 100% per send out); some participants considered further typing necessary and misclassification as negative was only sporadically seen. The classification of HLA‐B27neg samples as negative was less straightforward. Ten samples were correctly classified as such by 97% (82% to 100%) of the participants, whereas 64% (range 53% to 70%) of the participants classified the remaining eight samples as HLA‐B27neg. There was no significant prevalence of a particular HLA‐B allele among these eight “poor concordancy” samples as compared to the ten “good concordancy” samples. Inspection of the reactivity patterns of the individual MoAb with HLA‐B27neg samples revealed that ABC‐m3 showed very little cross‐reactivity apart from its well‐known cross‐reactivity with HLA‐B7, whereas the cross‐reactivity patterns of GS145.2 and FD705 were more extensive. The small sample size (n = 18) and the distribution of HLA‐B alleles other than HLA‐B27 did not allow assignment of specificities to these cross‐reactions. Finally, we showed that standardized interpretation of the combined results of two anti‐HLA‐B27 MoAb reduced the frequency of false‐positive conclusions on HLA‐B27neg samples. In this series, the lowest frequency of false‐positive assignments was observed with the combination of the FD705 and ABC‐m3 MoAb. Cytometry (Comm. Clin. Cytometry) 42:95–105, 2000. © 2000 Wiley‐Liss, Inc.