Folded proteins can be translocated across biological membranes via the Tat machinery. It has been shown in vitro that these Tat substrates can interact with membranes prior to translocation. Here we report a monolayer and infrared reflection-absorption spectroscopic (IRRAS) study of the initial states of this membrane interaction, the binding to a lipid monolayer at the air/water interface serving as a model for half of a biological membrane. Using the model Tat substrate HiPIP (high potential iron-sulfur protein) from Allochromatium vinosum, we found that the precursor preferentially interacts with monolayers of negatively charged phospholipids. The signal peptide is essential for the interaction of the precursor protein with the monolayer because the mature HiPIP protein showed no interaction with the lipid monolayer. However, the individual signal peptide interacted differently with the monolayer compared to the complete precursor protein. IRRA spectroscopy indicated that the individual signal peptide forms mainly aggregated β-sheet structures. This β-sheet formation did not occur for the signal peptide when being part of the full length precursor. In this case it adopted an α-helical structure upon membrane insertion. The importance of the signal peptide and the mature domain for the membrane interaction is discussed in terms of current ideas of Tat substrate-membrane interactions.