The discovery of cytokines expressed by T-helper 1 (Th1), Th2, Th17 and T-regulatory (Treg) cells has prompted speculation that these types of responses may exist in fish, arising early in vertebrate evolution. In this investigation, we cloned three zebrafish transcription factors, T-box expressed in T cells (t-bet), signal transducer and activator of transcription 6 (stat6) and fork-head box p3 (foxp3), in which two transcripts are present, that are important in the development of a number of these cell types. They were found within the zebrafish genome, using a synteny approach in the case of t-bet and foxp3. Multiple alignments of zebrafish t-bet, stat6 and foxp3 amino acids with known vertebrate homologues revealed regions of high conservation, subsequently identified to be protein domains important in the functioning of these transcription factors. The gene organizations of zebrafish t-bet and foxp3 were identical to those of the human genes, with the second foxp3 transcript lacking exons 5, 6, 7 and 8. Zebrafish stat6 (21 exons and 20 introns) was slightly different from the human gene, which contained 22 exons and 21 introns. Immunostimulation of zebrafish head kidney and spleen cells with phytohaemagglutinin, lipopolysaccharide or Poly I:C, showed a correlation between the expression of t-bet, stat6 and foxp3 with other genes involved in Th and Treg responses using quantitative PCR. These transcription factors, together with many of the cytokines that are expressed by different T-cell subtypes, will aid future investigations into the Th and Treg cell types that exist in teleosts.