We first used flow cytometry to screen plasma from 27 PAM-exposed and recently pregnant multigravidae for IgG antibodies capable of staining P. falciparum-IEs expressing VSAPAM Ricke et al., 2000) . We selected three donors (one parity 2 and two parity 3 women) with high VSAPAM-specific plasma antibody levels and used frozen peripheral blood mononuclear cells (PBMC) obtained 1 month post-partum. Memory B cells were immortalized with EBV in the presence of CpG oligonucleotides and allogeneic, irradiated PBMC as described (Traggiai et al., 2004) . A total of 5760 replicate cultures of 100 immortalized B cells per well were set up, and after 3 weeks the culture supernatants were screened for their capacity to stain erythrocytes infected with each of three P. falciparum lines. Two of the lines (FCR3-CSA and NF54-VAR2CSA) had been previously selected in vitro to express VSA PAM, characterized by reactivity with IgG from multiparous women and lack of reactivity with IgG from P. falciparum-exposed men ( Fig. 1) (Fried et al., 1998; Beeson et al., 1999; Ricke et al., 2000) . The third line (3D7-SM) was selected to express non-PAM-type VSA equally recognized by IgG from P. falciparum-exposed men and women ( Fig. 1) Jensen et al., 2004) . Supernatants from 105 of the polyclonal B-cell lines stained one or both of the VSAPAM-expressing lines. The frequency of VSAPAM-reactive polyclonal supernatants varied from 6/1920 [0.3% (95% confidence interval: 0.1-0.7%)] to 33/1344 [2.5% (1.8-3.4%)] in the three donors. These results suggest that the frequency of VSAPAM-specific B cells can be high (at least up to 1 in 4000 memory B cells) in recently pregnant multigravidae. The higher memory B-cell frequencies in the present study compared with earlier reports for PfEMP1 (Dorfman et al., 2005) and total P. falciparum antigens (Fievet et al., 1993; Migot et al., 1995) probably reflect the efficient method of B-cell immortalization employed here.