A biochemical pathway is the representation of a defined set of substrates, enzyme reactions and products linked together to generate an outcome beneficial to a living cell. Microbial genome sequence data are unparalleled resources for understanding cellular metabolism without the prior definitions imposed by classical biochemistry. Simple analysis of three well-studied biochemical pathways (the tricarboxylic acid cycle, pentose phosphate pathway and glycolysis) from the 17 publicly available microbial genomes has shown that these pathways may rarely occur as previously defined. Therefore, following whole-genome sequencing it has become necessary to redefine the "classical" biochemical steps leading from substrate to end-product for each pathway. Often, unique or alternative reactions appear to be required in order to maintain pathway functionality where expected enzyme reactions (as defined by the presence or absence of the corresponding genes) are "missing". Conversely, such enzymes may be accounted for by: (1) the presence of low sequence similarity or novel genes encoding enzymes performing the same or similar functions, (2) the presence of multienzyme proteins, (3) incorrectly assigned gene identities in genome databases, and (4) known enzyme functions that have yet to be correlated with a gene sequence. Most importantly, the presence of a gene sequence does not necessarily ensure that its corresponding enzyme is actually functional. This may be due to the presence of nonactive remnant genes, evolutionary pressures leading to loss of function, inactivating mutations, the lack of transcription/translation, and post-translational processing. Modifications at the gene and/or functional levels, as well as the possible use of alternative enzymes, must be considered when reconstructing biochemical pathways for fully sequenced microbial genomes.