T cells require two distinct signals for optimal activation. One is an antigen-specific signal and is provided by engagement of the T cell receptor (TCR). The second is an antigen-independent signal mediated by engagement of the T cell surface molecule CD28 with members of the B7 family. To endow CD28 molecules with antibody-type recognition, we have constructed chimeric single-chain antibody variable fragment (scFv)-CD28 molecules; following transfection of the genes encoding such constructs into the Jurkat human T cell line we show that they are stably expressed as functional cell surface receptors. These chimeric molecules have no apparent negative effects on the expression and signaling ability of the wild-type CD28 and TCR/CD3 molecules. When combined with signaling via the TCR/CD3 complex, these antigen-specific scFv-CD28 chimeric molecules provide signals similar to those elicited by the cross-linking of the unmodified CD28 molecules. Furthermore, the generation of double transfectants simultaneously expressing scFv-CD28 and scFv-CD3 zeta chimeras demonstrates that antigen-specific co-stimulatory signals can also synergize with signals mediated through chimeric CD3 zeta chains to secrete maximal levels of interleukin-2. Overall, our results suggest that optimal, predefined antigen-specific activation of T cells directed by the specificity of the scFv should be possible.