CD44, a cell adhesion molecule, exists in multiple isoforms that are generated by RNA alternative splicing. CD44 isoforms containing exon V6 (CD44 V6) have been associated with tumorigenesis and metastasis. We investigated the association between human B-cell activation and CD44 V6 isoform expression by analysing its expression in resting and mitogenically stimulated B cells. Results showed that resting B cells expressed the CD44 H (no variable exon) isoform alone. Activation of B cells [phorbol myristate acetate (PMA), surface immunoglobulin cross-linking alone or in the presence of interleukin-2 (IL-2)] induced CD44E (variable exon V8-10), R2 (VIO) and CD44 isoforms containing exons V6 and/or V7 (CD44 V6/V7). Epstein-Barr virus (EBV) infection of B cells, an alternative method of B-cell activation, induced the expression of CD44 E and R2 but not CD44 V6/V7. These results indicate that CD44 V6/V7 expression depends on the mode of activation. CD44 isoform expression was also investigated in a panel of EBV-negative and EBV-positive Burkitt's lymphoma (BL) B-cell lines. EBV-negative BL cells did not express CD44. In contrast, EBV-positive BL cells expressed CD44 H, R2 and E but not CD44 V6/V7 isoforms, suggesting an association between EBV infection and CD44 isoform induction. To determine directly the role of EBV in CD44 isoform induction, an EBV-negative BL cell line, BL30 (negative for all isoforms of CD44), BL30 infected in vitro with the EBNA-2-defective P3HR1 (BL30/P3HR1), and the wild-type B95-8 strain of EBV (BL30/B95-8) were examined. The parental BL30 cells infected with the wild-type EBV strain, but not with the P3HR-1 strain, expressed CD44 H, R2 and E isoforms, as seen in EBV-immortalized B cells. These studies suggest that (1) alternative splicing of CD44 isoforms is differentially regulated depending on the mode and state of cell activation, and that (2) the CD44 V6/V7 isoforms may represent B-cell activation antigens that are induced by mitogenic stimulation but not following EBV infection.