The need for improved adjuvants has continued in spite of new vaccination strategies using recombinant proteins and genetic (DNA vectored) vaccines. The Mycobacterium tuberculosis protein ESAT-6 has been shown in mice to be involved in the recall of long-lived immunity and protection in mice against tuberculosis. Thus, this protein could potentially act as a molecular adjuvant enhancing antigen-specific Type I immune responses. This study examined the hypothesis that Es could enhance Type I responses against a second antigen in mice when presented as a fusion with that antigen. This was tested using ESAT-6 fusions with two Mycoplasma hyopneumoniae surface antigens, P97 and P71, in mice either as recombinant proteins or as genetic vaccines. Control groups received no ESAT-6 sequences or no antigen or DNA. To accomplish this, gene sequences for the two proteins were identified and cloned with or without ESAT-6 sequences into expression or DNA vaccine vectors. This required modification of three TGA codons in the P71 gene sequence. Following immunization, serum IgG1 and IgG2a antibody levels and splenic lymphocyte antigen-specific blastogenic responses, and levels of IFN-gamma and IL-10 released were measured. The protein vaccination experiments revealed an elevated Th1 response with the ESAT-6 fusion vaccinates that was characterized by higher levels of IFN-gamma and suppression of IL-10 secretion from the sensitized, antigen-stimulated splenocytes. The P71 protein vaccinates with ESAT-6 sequences had significantly higher levels of IgG2a compared to non-ESAT-6 P71 vaccinates. The DNA vectored vaccination experiments gave similar results. Splenic lymphocytes from P71 vaccinates with ESAT-6 sequences secreted higher levels of IFN-gamma and lower levels of IL-10 when stimulated with P71 antigen compared to the non-ESAT-6 P71 vaccinates. In addition, the experiments with genetic constructs for P71 revealed that the IgG2a antibody levels were significantly higher with the P71 vaccinates receiving ESAT-6 sequences compared to the non-ESAT-6 P71 vaccinates. In conclusion, ESAT-6 can act as a molecular adjuvant in mice to enhance antigen-specific Type I immune responses to both recombinant protein vaccines as well as genetic vaccines.