CD28 is a cell surface molecule that mediates a costimulatory signal crucial for T cell proliferation and lymphokine production. The signal transduction mechanisms of CD28 are not well understood. Itk, a nonreceptor protein tyrosine kinase specifically expressed in T cells and mast cells, has been implicated in the CD28 signaling pathway because of reports that it becomes phosphorylated on tyrosines and associates with CD28 upon cross-linking of the cell surface molecule. To determine whether Itk plays a functional role in CD28 signaling, we compared T cells from Itk-deficient mice and control mice for their responses to CD28 costimulation. T cells defective in Itk were found to be fully competent to respond to costimulation. Whereas the CD3-mediated proliferative response was severely compromised in the absence of Itk, the calcineurin-independent CD28-mediated response was significantly elevated when compared with cells from control animals. The augmented proliferation was not due to increased production of interleukin-2. The results suggest that Itk has distinct roles in the CD3 versus the CD28 signaling pathways. By negatively regulating the amplitude of signaling upon CD28 costimulation, Itk may provide a means for modulating the outcome of T cell activation during development and during antigen-driven immune responses.