The HA of the Aichi/68 strain and the chimaeric HAs were used to identify the epitopes of the clones in the first set. The HAs of Yamanashi/77 and Fukuoka/85 and their chimaeric HAs were used to identify the epitopes of the clones in the second set. The HAs of Sydney/97 and Wyoming/2003 and their chimaeric HAs were used to identify the epitopes of the clones in the third set. Numerous FCM analyses were systematically performed, and the summary is shown in Fig. 4. The FCM results were judged to be positive, indicating that the Ab bound to the HA expressed on the cell surface, or negative, indicating that the Ab did not bind to the HA. All nine clones in group 1 did not bind to either A68/188B2 or A68/219D but did bind to the other seven chimaeric HAs. Therefore, we concluded that they bound to site B2/D. Since F035-015 of group 2 did not bind to A68/142A but did bind to the other eight chimaeric HAs, including A68/121A and A68/133A, we concluded that it bound to the C-terminal side of site A. On the other hand, F033-038 in group 3 did not bind to A68/133A, A68/142A or A68/154B1, but it did bind to the other six chimaeric HAs. Therefore, we concluded that it bound to site A/B1 (Fig. 4a). http://vir.sgmjournals.org Epitope mapping of neutralizing Abs against H3N2 virus Fig. 3. FCM analyses of wt and chimaeric HAs of five influenza strains. F49 is a mouse mAb that recognizes an epitope commonly present on HA of H3 subtype influenza viruses (Hay et al., 2001). The Fab-PP form of Ab was used for F033-161 (a), F032-072 (b) and F008- 007 (c). Since clones in groups 4 and 5 bound to the HAs of 1977– 1999 strains, those of Sydney/97 were also used in the FCM analysis in addition to Yamanashi/77 and Fukuoka/85. F008-039 in group 4 did not bind to Y77/50C1, F85/50C1 or S97/275C2 but did bind to all of the other Yamanashi/ 77, Fukuoka/85 and Sydney/97 chimaeric HAs tested. Although it bound to Y77/276C2 and F85/276C2, site C2 of these chimaeric HAs was changed from GTCSC (1977 and 1985 type) to GKCNS (1997 type), and F008-039 bound not only to Yamanashi/77 and Fukuoka/85 HAs but also to Sydney/97 HA. Thus, we concluded that F008-039 bound to site C1/C2. While the results from the analysis of Sydney/97 indicated that three clones of group 5 bound to site E, they bound to all of the chimaeric HAs of Yamanashi/77 and Fukuoka/85 including site E chimaera. However, the clones of group 5 bound to the HAs of the 1977–1999 strains. Therefore, if they bound to site E, they could bind to the HA with the sequence from the 78th to 83rd residues, GFQNEK (1977 and 1985 type) or GFQNKE (1997 type), but could not bind to either VFQNET (1968 type) or GFQNKK (2003 type) (Fig. 1). The reason why they bound to chimaeric HAs Y77/82E and F85/82E was that site E sequence had been changed in both chimaeras 329