Mycobacterium vaccae is of major pharmaceutical interest as an immunotherapeutic agent. Although M. vaccae 15483 ATCC(T) strain displays smooth and rough colonial morphologies on solid culture media, it is not known in which conditions M. vaccae switches from one colonial morphotype to the other or whether there are biological differences, especially immunological, between them. We have found that the change from a smooth to rough stable variant occurs spontaneously at 30 degrees C. The analysis of the composition of the cell wall in both variants showed that the smooth morphotype presents an extracellular material that has never previously been described and was identified as a long-chain saturated polyester that, interestingly, is not produced by the rough morphotype. Our results also indicate that this compound could be implicated in the spreading ability of smooth colonies. Proliferation, IFN-gamma and IL-12(p40) production by splenocyte cultures was significantly higher in mice immunised with the rough variant compared with those immunised with the smooth one. This latter finding suggests that the different colonial morphology of M. vaccae may affect the immunomodulatory effects induced from M. vaccae preparations.