Inhibitor studies and mutant analysis revealed a C(30) pathway via 4,4'-diapophytoene and 4,4'-diaponeurosporene to 4,4'-diaponeursoporene-4-oic acid esters related to staphyloxanthin in Halobacillus halophilus. Six genes may be involved in this biosynthetic pathway and could be found in two adjacent gene clusters. Two genes of this pathway could be functionally assigned by functional pathway complementation as a 4,4'-diapophytoene synthase and a 4,4'-diapophytoene desaturase gene. These genes were organized in two operons together with two putative oxidase genes, a glycosylase and an acyl transferase ortholog. Pigment mutants were obtained by chemical mutagenesis. Carotenoid analysis showed that a white mutant accumulated 4,4'-diapophytoene due to a block in desaturation. In a yellow mutant carotenogenesis was blocked at the stage of 4,4'-diaponeurosporene and in an orange mutant at the stage of 4,4'-diaponeurosporene-4-oic acid. The protective function of these pigments could be demonstrated for H. halophilus after inhibition of carotenoid synthesis by initiation of oxidative stress. A degree of oxidative stress which still allowed 50% growth of carotenogenic cells resulted in the death of the cells devoid of colored carotenoids.