T cell activation via CD2 requires interaction of CD2 with several signaling molecules. To investigate the structural requirements for an association of CD2 with the tyrosine phosphatase CD45 and the zeta chain of the T cell receptor (TCR)/CD3/zeta complex, we have expressed in mouse EL4 T cells a series of human CD2 chimeric and mutant proteins. Chimeric proteins in which the CD2 transmembrane and/or cytoplasmic domains were deleted or exchanged with analogous regions of CD4, CD28 or CD58 retained association with high levels of murine CD45 phosphatase activity, suggesting that the CD2 extracellular domain largely controls interaction with CD45. To a lesser extent, the cytoplasmic domain of CD2 was also shown to interact with CD45, as demonstrated by an increase in co-immunoprecipitated phosphatase activity observed following replacement of the CD58 cytoplasmic domain with that of CD2. In contrast, the cytoplasmic domain of CD2 was found to be responsible for the majority of CD2 interaction with the zeta chain of the TCR/CD3/zeta complex. Deletion of the CD2 cytoplasmic domain, excluding the first three amino acids, removed virtually all CD2 associated zeta chain and approximately sevenfold higher levels of zeta chain were found in association with a CD58/58/2 chimera than with control human CD58 wild type. This study suggests that the CD2 extracellular and intracellular domains are differentially involved in regulating T cell activation through interaction with the tyrosine phosphatase CD45 and the zeta chain of the TCR/CD3/zeta complex.