CD45, a transmembrane protein tyrosine phosphatase (PTP), can either positively or negatively regulate Src-family protein tyrosine kinase (PTK) activity in vivo. It is proposed that TCR-initiated signaling requires the segregation of PTP activities from the engaged TCR, based upon the differential membrane compartmentalization on the T cell surface. To test the importance of CD45 exclusion from lipid microdomains for proper TCR signaling, a chimeric molecule was generated by fusing the CD45 cytoplasmic region, which contains the PTP domains, to the amino-terminal 12 amino acids of Lck, which target Lck to lipid microdomains. Using 3A9 T lymphocyte hybridoma (3A9H) cells whose TCR recognizes hen egg-white lysozyme (HEL), Lck-CD45 expression resulted in its targeting to lipid microdomains. The 3A9H cells expressing Lck-CD45 were reduced in their responses to HEL or co-cross-linking of CD3 and CD4, as assessed by IL-2 production and Ca(2+) mobilization. Src-family PTK activity associated with lipid microdomains was also decreased. These results suggest that the segregation of CD45 from proximal TCR signaling components is necessary for TCR signaling and that the targeting of CD45 PTP activity to lipid microdomains on the T cell surface results in decreased sensitivity of TCR-mediated signaling.