In the cyanobacterium Spirulina platensis, the desaturation process is carried out by three desaturases: the Delta(9), Delta(12) and Delta(6) desaturases, encoded by desC, desA and desD, respectively. The Delta(6) desaturase is responsible for the catalysis of linoleic acid, yielding gamma-linolenic acid (18:3(Delta9,12,6)), the end-product of the process. In this study, the desD gene was expressed in Escherichia coli using a pTrcHisA expression system. In order to identify the amino acid residues involved in the enzymatic activity, a sequence comparison was performed using various organisms. The alignment revealed three conserved histidine clusters, a number of conserved residues among all listed organisms and a few conserved residues among cyanobacterial species possibly involved in the desaturation activity. A series of site-directed mutations were generated in the desD gene to evaluate the role of these residues vis-a-vis the enzyme function. This approach revealed that: (1) H313 is involved in the regioselectivity of the enzyme, (2) the three histidine clusters together with H313, H315, D138 and E140 are required for enzymatic activity, most likely as providers of the catalytic Fe center and (3) W294 is also essential for the activity of Delta(6) desaturase, possibly by forming part of the substrate-binding pocket.