The role of aspartate-84, an invariant residue in the active site cleft of Escherichia coli porphobilinogen deaminase, has been investigated by site-directed mutagenesis. Substitution of aspartate-84 by glutamate results in an enzyme that retains less than 1% of its activity and which can form highly stable enzyme-intermediate complexes. Substitution of aspartate-84 by either alanine or asparagine, however, results in proteins unable to catalyze the formation of preuroporphyrinogen but which, nevertheless, appear able to assemble the dipyrromethane cofactor. The mechanisms of the tetramerization reaction and cofactor assembly are discussed.