The desaturation reactions of C(30) carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C(40) phytoene to some extend but exhibited a high affinity to zeta-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C(40) carotenes revealed that this desaturase can also convert C(30) diapophytoene. Other homologous bacterial C(40) carotene desaturases could also utilize C(30) substrates, including one type of zeta-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C(30) carotenogenic pathway is determined by this initial enzyme which is highly homologous to C(40) phytoene synthases.