Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis, a disease affecting sheep and goats. Phospholipase D (Pld), a major virulence determinant of C. pseudotuberculosis, is believed to play a critical role in dissemination of bacteria from the site of infection to the lymph nodes. Although the pld gene has been studied for some time, it is only recently that it has been identified as being down-regulated following heat shock from 37 to 43 degrees C. To gain insights into the mechanisms of Pld action, this study investigated how it was regulated under varying environmental conditions. Studies measuring pld mRNA levels or utilizing a reporter construct containing the pld promoter upstream of a gfp gene were performed. These showed that pld was upregulated in a cell-density-dependent manner, was regulated by heat shock at all cell-culture densities, and was highly expressed in a tissue-culture macrophage-infection model. Finally, the expression of Pld by intracellular C. pseudotuberculosis was shown to play a small but significant role in the reduction of macrophage viability following infection. This study demonstrates that the regulation of C. pseudotuberculosis pld is complex. This regulatory complexity may play an important role in allowing the pathogen to successfully adapt to the changing host environment during infection, migration, establishment and disease progression.