CD44 expression and the functional capacity for CD44-dependent binding of hyaluronic acid (HA) were analyzed on unstimulated B cells and on B cells stimulated with a variety of polyclonal B cell activators. Whereas essentially all LPS-activated and anti-IgD-dextran-activated B cells and a subpopulation of IL-5-activated B cells expressed increased levels of cell surface CD44 relative to unstimulated B cells, only IL-5-activated CD44hi B cells constitutively bound to FITC-conjugated hyaluronic acid (FITC-HA). Preincubation of LPS or anti-IgD-dextran-activated B cells with the CD44-specific mAb IRAWB14.4 (IRA) induced a high degree of FITC-HA binding in these populations; preincubation of unstimulated B cells with this CD44-specific mAb induced minimal FITC-HA binding. In contrast, preincubation with mAb IRA failed to induce FITC-HA binding by the IL-5-activated CD44lo B cell subset. Neither the amount of constitutive FITC-HA binding nor the level of IRA-inducible FITC-HA binding correlated simply with the overall level of CD44 expressed by the different B cell populations. Biochemical analysis of immunoprecipitated CD44 molecules revealed that relative to CD44 isolated from all other populations examined, CD44 isolated from IL-5-activated B cells was of a lower molecular weight. Treatment with N-Glycanase eliminated this observed difference in molecular weight, indicating that it reflected differences in N-glycosylation of CD44 on activated B cells. Polymerase chain reaction analysis of amplified cDNA showed that each B cell population expressed a common dominant CD44 mRNA. These findings suggest that post-translational modification of CD44 and/or differential association of CD44 with other cellular components plays a critical role in activation-specific ligand binding by CD44.