CD40 and CD40 ligand (CD40L) form one of most important receptor-ligand pairs that dock during T-B cell interactions as part of T-dependent antibody responses. It has been reported that among other cell types, B cells can express CD40L. Here we show that a large proportion of mouse B cells express CD40L in their cytoplasm, but not on the surface and that this is readily released as a soluble molecule. Thus, in their resting state up to 50% of mouse B cells express CD40L within their cytoplasm and both the proportion of cells expressing and the amount of CD40L is increased by signaling through immunoglobulin (Ig) or CD38. In contrast, T cell-derived signals such as CD40L (anti-CD40) or Th2-type cytokines cause a decrease in CD40L expression that is related to a release of a soluble form of the molecule from the cell. Supernatants from B cells activated with anti-Ig and anti-CD40 contain CD40L in a variety of forms (18 kDa, 33 kDa and 66 kDa) that are readily detectable by immunoprecipitation with CD40-Fc gamma fusion protein (CD40-Ig) followed by Western blotting with anti-CD40L antibody (MR1). The 33-kDa species is distinct from the 39-kDa membrane-bound molecule found in activated T cells or in resting B cells and appears to be a novel soluble form of CD40L. Inhibition of T cell-independent in vitro stimulation of B cells with CD40-Ig or anti-CD40L suggests that the B cell-derived soluble CD40L or CD40L expressed on the B cell surface can play a positive role in B cell proliferation.