In the present study we tested whether the forced expression of the CD3 zeta chain within detergent-resistant, glycosphingolipid-enriched membrane microdomains (GEMs) will result in a constitutively activated phenotype in human T cells, To this aim, a monomeric recombinant protein (LckSH4-CD3 zeta), containing the intracellular part of human CD3 zeta chain fused to N-terminal double-acylation motif (SH4 domain) of protein tyrosine kinase Lck, was expressed in Jurkat human T lymphoid cell line and its Lck-negative mutant, J.CaM1.6. The Lck SH4 domain indeed predominantly targeted the chimeric protein into GEMs. In transfectants derived from wild-type Jurkat cells, but not in those derived from the Lck-deficient mutant, the LckSH4-CD3 zeta protein was constitutively tyrosine-phosphorylated. Tyrosine phosphorylation of a major Jurkat cell phosphoprotein (pp85) was diminished in the transfectants. However, the transfectants did not exhibit any features of constitutively activated T cells, and their responses to anti-CD3 treatment were very similar to the wild-type Jurkat cells, Thus, the constitutive expression of this form of CD3 zeta chain in GEMs is not sufficient for eliciting an activated state in the Jurkat cells.