In silico subtractive/differential genome analysis is a powerful approach for identifying genus- or species-specific genes, or groups of genes that are responsible for a unique phenotype. By this method, one searches for genes present in one group of bacteria and absent in another group. A software package has been developed, named FindTarget, that has a user-friendly web interface to facilitate differential genome analysis. The user chooses the genomes to compare, the similarity criteria and the thresholds to decide if a gene has a counterpart in another genome. The searches are based on BLASTP comparisons of proteomes. FindTarget also includes access to sequences, coloured multiple alignments, phylogenetic trees of conserved proteins and links to public annotated databases which provide a means for validation of the results. To illustrate this approach, a FindTarget search for genes putatively involved in the specificity of cell envelope synthesis of Gram-negative bacteria is presented. The results show that most of the identified genes are clearly involved in cell wall processes, underlining the power of such an approach in general and that of FindTarget in particular.