BACKGROUND: Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable losses for herd owners. Phospholipase D (PLD) is a potent exotoxin produced by C. pseudotuberculosis and it has been considered as the major virulence factor for this bacterium, possibly contributing to the spread of the bacteria from the initial site of infection to secondary sites within the host. Heat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. OBJECTIVES: The aim of this study was the cloning and expression of the PLD and HSP60 genes of C. pseudotuberculosis, used subsequently to evaluate the protectivity of these recombinant proteins for vaccine development against this bacterium. METHODS: PLD and HSP60 genes were cloned into pMAL-c2X vector and recombinant plasmids construct was transformed to DH5 strain of E. coli. Expression of the proteins was shown by SDS-PAGE and accuracy of the cloned genes was confirmed by nucleotide sequence analysis. RESULTS: The transformed E. coli strain DH5 expressed PLD and HSP60 proteins effectively. The expressed fusion protein was found almost entirely in the soluble form. CONCLUSIONS: In the following studies the immunogenicity and protectivity of these recombinant proteins against C. pseudotuberculosis infections can be assessed.