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The current UK anthrax vaccine is an alum precipitate prepared from static culture filtrate of the avirulent, unencapsulated Sterne strain of Bacillus anthracis. Protective antigen (PA) is regarded as the major immunogen in the vaccine and production conditions are intended to maximize the PA content. However, the precise composition of the vaccine is unknown and there are concerns that the observed side effects of vaccination may be caused by residual enzymatically active toxin components. Two-dimensional gel electrophoresis (2DGE) was used to define the protein components of the current UK anthrax vaccine. Consistency of composition was assessed by examining batches spanning 14 years of vaccine production. The reproducibility of the 2DGE technique was assessed by repeated analysis of selected vaccine batches. For two recently produced batches, between 86.7 and 88.8% of the spots could be matched. However, for one older batch, reproducibility of the spot pattern was considerably less, with a mean similarity of 53.4%. This difference may be explained by a change in production or because of decay during storage. Variation between the recently produced batches ranged from 72.9 to 84.3%, whereas the similarity between these and old batches was comparatively low at between 30 and 59%. Our results demonstrate that, as expected, the major antigen present in the vaccine is PA. The 83 and 63 kDa species are dominant but there are numerous lower molecular weight fragments resulting from proteolytic cleavage. In addition, we have established the presence of the toxin components, oedema factor and lethal factor, and S-layer proteins, EA1 and SAP. Mass spectrometry has also enabled us to identify several bacterial cell-derived proteins present in the vaccine, including PA, enolase, fructose-bisphosphate aldolase, nucleoside diphosphate kinase and a 60 kDa heat shock protein. The use of proteomics can provide useful information on the antigenic make up of this vaccine and the consistency of vaccine production.