Bacterial lipoproteins are characterized by fatty acids, derived from membrane phospholipids, which are covalently attached to their amino terminus via posttranslational modification in the cytoplasmic membrane. Here, I describe the detection of one of the intermediate forms of lipoprotein, diacylglyceryl-prolipoprotein, using 3H-palmitate labeling and inhibition of signal peptidase II (Lsp) by globomycin and detection by fluorography.