Fatty acid desaturases (FADs) represent a class of oxygen-dependent enzymes that dehydrogenate C–C bonds in fatty acids (FAs) producing unsaturated C=C bonds that markedly change the properties of lipid membranes, which is critical for protein and lipid diffusion, perception and transduction of environmental signals, cell division, etc. Membrane-located FADs (acyl-lipid FADs of plants and bacteria, as well as animal acyl-coenzyme A (CoA) FADs) are highly conserved from viruses to human. These enzymes are highly specific towards their acyl substrates, as well as towards the position and geometric configuration of the newly introduced cis double bonds. The regiospecificity of soluble Acyl-Carrier-Protein (ACP) Δ9-FADs was determined relative to the carboxyl end of the FA. Similar regiospecificity was suggested for acyl-lipid and acyl-CoA FADs Δ9-FADs. It was previously thought that acyl-lipid Δ12-FADs (also known as ω6-FADs) also count from the carboxyl terminus of an FA. However, heterologous expression and supplementation of model yeast or cyanobacterial strains with exogenous monounsaturated odd- and even-chain FAs revealed that plant and cyanobacterial acyl-lipid Δ12-FADs use neither end of the FA (Δ or ω) as a counting reference point; but count three carbons toward the methyl end from an existing double bond in the monoene precursors irrespective of FA chain length. Δ6-FADs appeared to ‘count’ from the carboxyl terminus. ω3-FADs that contribute to unsaturation of C16–C18 FAs of membrane lipids, in fact, are Δ15-FADs that also ‘count’ from the C-terminus of acyl chains. The exact knowledge of counting order is crucial for understanding the modes of FADs activities and for further construction of biochemical pathways for biosynthesis of polyunsaturated FAs.