The lipid modification of membrane proteins was investigated in Acholeplasma laidlawii by metabolic labeling and by chemical analysis. A S-glycerylcysteine residue was identified from membrane proteins and we reported the strong preference for saturated acyl chains into the lipid modification. Differential release of fatty acids revealed a ratio [(O-ester- + amide-bound acyl chains)/O-ester-linked chains] close to 1.1 which suggests the involvement of only two O-ester linked fatty acids in the acylation process. Present data indicate that acyl proteins in A. laidlawii are true lipoproteins (mainly diacylated) probably processed by a mechanism analogous to that described for eubacteria and other mycoplasmas.