Cells of the obligate halophile Halobacterium cutirubrum were grown to the stationary phase in the presence of 32P-orthophosphate, 35S-sulfate, or 14C-labelled precursors (acetate, malonate, mevalonate, glycerol). 32P was incorporated into the phosphatide components in proportion to their relative concentrations, thus: phosphatidyl glycerophosphate (diether analogue) > unidentified minor phosphatide > phosphatidyl glycerol (diether analogue). 35S-Sulfate was incorporated mostly into the glycolipid sulfate ester component, but small amounts of 35S also appeared in a more polar sulfolipid and in a minor unidentified sulfolipid.14C-Mevalonate was the most efficient precursor for the phytanyl chains, followed in decreasing order by acetate and glycerol. Only traces of 14C from the malonate precursor were incorporated, and these appeared entirely in the phytanyl groups. A considerable proportion of 14C from the glycerol precursor was found in the glycerol and sugar moieties of the lipids. Little or no labelling of the water-soluble moieties of the lipids was observed with the other carbon precursors.Only traces of 14C-labelled fatty acids were detected with any of the 14C precursors; with acetate, in particular, only 0.3% of the 14C in the total lipids was found in fatty acids. It is concluded that the predominant biosynthetic route for the lipid chains is the mevalonate pathway for synthesis of isoprenoid groups, and that the malonyl-CoA pathway for synthesis of fatty acids is operating at a very low level of activity.