Based on recent analytical and enzymological studies, a topological model for the role of alpha-D-mannosyl-(1-->3)-alpha-D-mannosyl-(1-->3)-diacylglycerol (Man(2)-DAG) as a lipid anchor precursor and mannosylphosphorylundecaprenol (Man-P-Und) as a mannosyl donor in the assembly of a membrane-associated lipomannan (LM) in Micrococcus luteus has been proposed. In this study, a [(3)H]mannose-suicide selection procedure has been used to identify temperature-sensitive (ts) mutants defective in LM assembly. Two micrococcal mutants with abnormal levels of Man(2)-DAG and LM at the nonpermissive temperature (37 degrees C), mms1 and mms2, have been isolated and characterized. In vivo and in vitro biochemical assays indicate that mms1 cells have a defect in the mannosyltransferase catalyzing the conversion of Man-DAG to Man(2)-DAG, and mms2 has a temperature-sensitive defect in the synthesis of Man-P-Und. Because mms1 cells are depleted of endogenous Man(2)-DAG, membranes from this mutant efficiently converted purified, exogenous [(3)H]Man(2)-DAG to [(3)H]LM by a Man-P-Und-dependent process. An obligatory role for Man-P-Und as a mannosyl donor in the elongation process was also demonstrated by showing that the conversion of exogenous [(3)H]Man(2)-DAG to [(3)H]LM by membranes from mms1 cells in the presence of GDP-Man was inhibited by amphomycin. In addition, consistent with Man(2)-DAG serving as a lipid anchor precursor for LM assembly, endogenous, prelabeled [(3)H]Man(2)-DAG was converted to [(3)H]LM when membranes from mms2 cells were incubated with purified, exogenous Man-P-Und. These studies provide the first direct proof for the role of Man(2)-DAG as the lipid anchor precursor for LM, and suggest that Man(2)-DAG may be essential for the normal growth of M. luteus cells.