CD28 costimulatory signals are required for T-cell proliferation and lymphokine production. In this work, the functional conservation of CD28 was studied in avian gammadelta T cells. The avian CD28 molecule is expressed on all alphabeta T cells and is capable of giving a costimulatory signal. Most peripheral gammadelta T cells are CD28 negative; however, we identified a CD28-positive gammadelta T-cell subset from peripheral blood comprising about 12% of gammadelta T cells. The peripheral CD28+ gammadelta T-cell subset included all CD8+ gammadelta T cells known to be a responding subset during activation. After polyclonal activation, the frequency of CD28+ gammadelta T cells was increased and the activation also up-regulated CD5, CD25 and major histocompatibility complex (MHC) class II molecules. These changes were detected after both polyclonal and antigen-specific T-cell activation. In addition, we also showed that CD28 can give a costimulatory signal to gammadelta T cells and that this signal leads to up-regulation of IL-2 and bcl-x transcripts. These results indicate that the function of CD28 is evolutionarily conserved and can already be detected in avian gammadelta T cells.