We have used the technique of subtractive hybridization to identify a T cell gene selectively expressed during activation via the antigen-receptor pathway. This gene, termed TCA3 (for T cell activation) encodes a mRNA which is expressed following concanavalin A (Con A) activation of T cell clones at levels of approximately 1% total poly(A)-containing mRNA. The cDNA isolate, termed TCA3.0, is 512 bases in length excluding poly(A) and encodes a predicted 92-amino acid protein having the characteristics of a secreted polypeptide of approximately 69 amino acids. The genomic organizations of TCA3 was determined for two lambda phage clones and was found to be a single copy gene containing at least three exons dispersed over less than 4.7 kb. The temporal appearance of TCA3 mRNA in response to several activating agents was examined. It is not transcribed in response to interleukin 2 stimulation, but is transcribed in response to either antigen or Con A stimulation and can be detected as early as 1 hr poststimulation. Expression TCA3 in response to Con A is blocked by cyclosporin A treatment. The combined data suggest that TCA3 may represent a new lymphokine.