T lymphocyte activation requires a combination of signals, one of which is provided by interaction between CD28 and its ligands on antigen presenting cells. Although CD28-like sequences have been identified in a few teleosts, the function of fish CD28 is virtually unknown. In this study, we cloned and analyzed a CD28 gene, CsCD28, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsCD28 contains 229 residues and shares 20.2%-40.3% overall sequence identities with known fish CD28 sequences. CsCD28 possesses structural features conserved in mammalian and teleost CD28, which include the MYPPPY motif in the extracellular immunoglobulin-like domain and the YXN motif in the cytoplasmic domain. The CsCD28 gene is 2746 bp and composed of four exons and three introns, which in organization resemble those of mammalian and trout CD28. Quantitative real time RT-PCR analysis showed that CsCD28 expression occurred predominately in kidney, spleen, gut, and gill. CsCD28 is localized on the surface of head kidney lymphocytes, and antibody ligation of CsCD28 induced significant levels of cellular proliferation. Taken together, these results indicate that CsCD28 is similar to mammalian CD28 in genetic and protein structures and possibly plays a role in T cell activation.